Bioartificial ultrafiltration device and methods related thereto

ABSTRACT

Bioartificial ultrafiltration devices comprising a scaffold comprising a population of cells enclosed in a matrix and disposed adjacent a plurality of channels are provided. The population of cells provides molecules such as therapeutic molecules to a subject in need thereof and is supported by the nutrients filtered in an ultrafiltrate from the blood of the subject. The plurality of channels in the scaffold facilitate the transportation of the ultrafiltrate and exchange of molecules between the ultrafiltrate and the population of cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/304,758 filed Mar. 7, 2016 and to U.S. Provisional Application No.62/328,298 filed Apr. 27, 2016, which are both herein incorporated byreference in their entirety.

INTRODUCTION

Type 1 diabetes (T1D) results from autoimmune destruction of theinsulin-producing β-cells within the pancreatic islets of Langerhans.Islet transplantation by direct infusion of cadaveric islets into theportal vein of the recipient's liver offers a non-invasive cure forpatients with T1D mellitus 1. However, donor availability, poorengraftment, and side effects from global immunosuppression remain asobstacles for wider application of this approach. Moreover, up to 60% ofthe infused islets become nonviable within a few days after surgicaldelivery and the long-term insulin independence is frequently lost by 5years of transplantation. The activation of innate and the adaptiveimmune responses are among the main causes of islet graft failure. Theidea of encapsulating islets has generated tremendous interest. However,there is a need for improved devices and methods for providingencapsulated islets that maintain function and are protected from thepatient's immune system.

SUMMARY

Bioartificial ultrafiltration devices for transplantation of cells in asubject are disclosed. These devices include a scaffold thatencapsulates a population of cells while providing a plurality ofchannels adjacent the population of cells. In certain embodiments, aplanar scaffold for facilitating exchange of molecules between aplurality of channels and cells adjacent the plurality of channels isdisclosed. The planar scaffold may include a solid planar substratecomprising a first surface and a second surface; a void in the solidplanar substrate, wherein the void extends from the first surface to thesecond surface; a matrix disposed in the void and extending from thefirst surface to the second surface, the matrix comprising: a pluralityof channels extending from the first surface to the second surface, anda population of cells adjacent the plurality of channels.

In certain embodiments, the solid substrate has a thickness of 0.1 mm-10mm, such as, 0.5 mm-5 mm, or 0.5 mm-3 mm and the first and secondsurfaces each have a surface area of 1 cm²-100 cm². The matrix may havea surface area of 1 mm²-10,000 mm², e.g., 1 mm²-5000 mm², 1 mm²-1000mm², 1 mm²-100 mm². In certain embodiments, the matrix comprises up to25,000 channels. In certain embodiments, the channels comprise a widthof 5 micron-1000 micron, e.g., a width of 10 micron-200 micron. Incertain embodiments, the channels are circular and the width refers todiameter of the channels. In certain embodiments, the channels arerectangular. In certain embodiments, the length of the channels rangesfrom 100 micron to 1000 micron. In certain embodiments, a population ofcells is separated from an adjacent channel by a distance of up to 500microns or less, such as, 400 microns or less, or 300 microns or less.The population of cells may be insulin secreting cells. The populationof cells may be pancreatic cells isolated from pancreatic islets,pancreatic islet cells, pancreatic beta cells, pancreatic islet cells,or cells in a pancreatic islet. Insulin secreting cells may be generatedby differentiation of stem cells, such as, induced pluripotent stemcells (iPSCs).

The scaffold may include a semipermeable ultrafiltration membranedisposed on a first surface of the scaffold and covering the matrix onthe first surface. In certain embodiments, a semipermeableultrafiltration membrane may be disposed on a second surface of thescaffold and may be covering the matrix on the second surface. Thesemipermeable ultrafiltration membrane may be sized to immunoisolate thecells encapsulated in the matrix.

The planar scaffold may be used for culturing the population of cells,for example, for maintaining viability of the cells. The planar scaffoldmay be used for manufacturing a bioartificial ultrafiltration device asdescribed herein.

In certain embodiments, the bioartificial ultrafiltration device mayinclude a planar scaffold comprising a matrix comprising a population ofcells and a plurality of channels adjacent to the population of cells,wherein the channels extend from a first surface to a second surface ofthe planar scaffold; a first semipermeable ultrafiltration membranedisposed on the first surface of the planar scaffold; a firstcompartment adjacent to the first surface of the planar scaffold and influidic communication with the planar scaffold via the firstsemipermeable ultrafiltration membrane and comprising an inlet and anoutlet; a second compartment adjacent to the second surface of theplanar scaffold and comprising an outlet, wherein the firstsemipermeable ultrafiltration membrane comprises a plurality of poreshaving a width in the range of 5 nm-5 micron, wherein the firstsemipermeable ultrafiltration membrane allows transport of ultrafiltratefrom the first compartment to the plurality of channels and wherein theultrafiltrate traverses from the plurality of channels into the secondcompartment.

In certain embodiments, the device may further include a secondsemipermeable ultrafiltration membrane disposed on the second surface ofthe planar scaffold and wherein the ultrafiltrate traverses from theplurality of channels across the second semipermeable ultrafiltrationmembrane into the second compartment. The second semipermeableultrafiltration membrane may include a plurality of pores having a widthin the range of 5 nm-5 micron. In certain embodiments, the first andsecond semipermeable ultrafiltration membranes may include a pluralityof pores having a width in the range the range of 0.1 microns-2 microns.

In certain embodiments, the second semipermeable ultrafiltrationmembrane comprises a plurality of pores having a width larger than thewidth of the plurality of pores in the first semipermeableultrafiltration membrane. In certain embodiments, the secondsemipermeable ultrafiltration membrane comprises a plurality of poreshaving a width smaller than the width of the plurality of pores in thefirst semipermeable ultrafiltration membrane.

In certain embodiments, the inlet of the first compartment is attachableto a conduit for connection to a blood vessel of a subject. In certainexamples, this blood vessel may be an artery of the subject in whom thedevice is transplanted.

In certain embodiments, the outlet of the first compartment isattachable to a conduit for connection to a blood vessel of a subject.In certain examples, this blood vessel may be a vein or an artery of thesubject. In certain cases, the outlet may be connect to an artery whichmay be the same artery as or different artery than the artery to whichthe inlet is connected.

In certain examples, the first compartment comprises a plurality ofoutlets that are each attachable to conduits for connection to (i) aplurality of different blood vessels of a subject or (ii) a plurality ofconnection sites on a single blood vessel.

In certain examples, the outlet of the second compartment is attachableto a conduit for connection to a blood vessel or a body cavity of asubject. In certain cases, the outlet of the second compartment providesthe ultrafiltrate to one or more blood vessels or body cavity of thesubject. In certain cases, the outlet of the second compartment isattachable to a tubing for connection to one or more veins of thesubject. In other cases, the outlet of the second compartment isattachable to a tubing for connection to one or more arteries of thesubject. In some cases, the outlet of the second compartment isattachable to an analyte analysis device. In yet other embodiments, thesecond compartment comprises a plurality of outlets for providing theultrafiltrate to at least one blood vessel or a body cavity of thesubject. In some cases, the second compartment comprises a plurality ofoutlets for providing the ultrafiltrate to an analyte analysis device.

In another embodiment, a bioartificial ultrafiltration device mayinclude a first planar scaffold and a second planar scaffold eachcomprising a matrix comprising a population of cells and a plurality ofchannels adjacent the population of cells, wherein the channels extendfrom a first surface to a second surface of each of the planarscaffolds; a first semipermeable ultrafiltration membrane disposed onthe first surface of the first and second planar scaffolds; a firstcompartment adjacent to and sandwiched between the first surface of thefirst and second planar scaffolds and comprising an inlet and an outlet,wherein the first semipermeable ultrafiltration membrane allowstransport of ultrafiltrate from the first compartment to the scaffolds;a second compartment adjacent to the second surface of the first planarscaffold and comprising an outlet; a third compartment adjacent to thesecond surface of the second planar scaffold and comprising an outlet,wherein the first semipermeable ultrafiltration membrane comprises aplurality of pores having a width in the range of 5 nm-5 micron, andwherein the ultrafiltrate traverses from the plurality of channels inthe scaffolds into the second compartment and the third compartment. Incertain embodiments, the second semipermeable ultrafiltration membranemay include a plurality of pores having a width in the range of 5 nm-5micron and wherein the second semipermeable ultrafiltration membrane isdisposed on the second surface of the first and second planar scaffoldsand wherein the ultrafiltrate traverses from the plurality of channelsin the scaffolds into the second compartment and the third compartmentvia the second semipermeable ultrafiltration membrane.

In certain embodiments, the first semipermeable ultrafiltration membranecomprises a plurality of pores having a width in the range the range of0.1 microns-2 microns. In certain embodiments, the second semipermeableultrafiltration membrane comprises a plurality of pores having a widthin the range the range of 0.1 microns-2 microns. In certain embodiments,the second semipermeable ultrafiltration membrane comprises a pluralityof pores having a width larger than the width of the plurality of poresin the first semipermeable ultrafiltration membrane. In certainembodiments, the second semipermeable ultrafiltration membrane comprisesa plurality of pores having a width smaller than the width of theplurality of pores in the first semipermeable ultrafiltration membrane.

In certain embodiments, the inlet of the first compartment isconnectable to a conduit for connection to an artery of a subject. Incertain embodiments, the outlet of the first compartment is connectableto a conduit for connection to an artery or a vein of a subject. Incertain embodiments, the outlet of the second compartment is connectableto a conduit (e.g., a tubing) for connection to at least one bloodvessel of a subject and/or to an analyte analysis device. In certainembodiments, the outlet of the third compartment is connectable to atubing for connection to at least a blood vessel of a subject and/or toan analyte analysis device. In certain embodiments, the outlet of thethird compartment is connectable to a tubing for connection to at leasta vein of a subject and/or to an analyte analysis device. In certainembodiments, the outlet of the second compartment and the outlet of thethird compartment are connected to a single tubing for connection to atleast a blood vessel of a subject and/or to an analyte analysis device.

In certain embodiments, the plurality of pores in the firstsemipermeable membrane have a width in the range of 0.2 μm-0.5 μm andthe plurality of pores in the second semipermeable membrane have a widthin the range of 0.2 μm-0.5 μm. In certain embodiments, the thickness ofthe first semipermeable ultrafiltration membrane is in the range of 0.1micron-1000 micron and the thickness of the second semipermeableultrafiltration membrane is in the range of 0.1 micron-1000 micron.

In certain embodiments, the surface area of the first and secondsemipermeable ultrafiltration membrane is in the range of 1 cm²-100 cm².In certain embodiments, the surface area of the first and secondsemipermeable ultrafiltration membrane is in the range 15 cm²-30 cm².

As noted herein, the plurality of pores may be circular in shape andwherein the width refers to diameter of the pores. In some embodiments,the plurality of pores are slit-shaped and where the length of the poresis in the range of 0.1 micron-5 micron, e.g., 1 μm-3 μm.

The cells in the device may be insulin producing cells. For example, theinsulin producing cells are derived from differentiation of stem cellsor are pancreatic cells isolated from pancreatic islets. The cells maybe autologous, allogenic, or xenogenic to the subject in whom the devicewill be transplanted.

A further embodiment of an bioartificial ultrafiltration devicedisclosed herein includes a planar scaffold comprising a matrixcomprising a population of cells and a plurality of channels adjacentthe population of cells, wherein the channels extend from a firstsurface to a second surface of the planar scaffold; a firstsemipermeable ultrafiltration membrane disposed on the first surface anda second semipermeable ultrafiltration membrane disposed on the secondsurface of the planar scaffold; a first compartment comprising a firstinlet and a first outlet, wherein the first compartment is adjacent tothe first surface of the planar scaffold; a second compartmentcomprising a second inlet and a second outlet, wherein the secondcompartment is adjacent to the second surface of the planar scaffold,wherein the first inlet is configured for connection to an artery of asubject and the first outlet is connected to the second inlet of thesecond compartment, wherein the second outlet of the second compartmentis configured for connection to a vein of the subject, wherein thesemipermeable ultrafiltration membranes comprise a plurality of poreshaving a width in the range of 5 nm-5 micron, wherein the firstsemipermeable ultrafiltration membrane allows transport of ultrafiltratefrom the first compartment to the scaffold and the second semipermeableultrafiltration membrane allows transport of the ultrafiltrate from theplurality of channels in the scaffold into the second compartment. Thecells present in the device may insulin producing cells as describedherein. In certain cases, the plurality of pores have a width in therange of 0.2 μm-0.5 μm.

In some embodiments, the plurality of pores in the second semipermeableultrafiltration membrane have a width larger than the width of theplurality of pores in the first semipermeable ultrafiltration membrane.In some embodiments, the plurality of pores in the second semipermeableultrafiltration membrane have a width smaller than the width of theplurality of pores in the first semipermeable ultrafiltration membrane.

In some embodiments, the thickness of the semipermeable ultrafiltrationmembranes is in the range of 0.1 micron-1000 micron, e.g., 200 μm-1000μm. The surface area of the semipermeable ultrafiltration membrane is inthe range of 1 cm²-100 cm², e.g., 15 cm²-30 cm².

As noted herein, the plurality of pores may be circular in shape andwherein the width refers to diameter of the pores.

In some embodiments, the plurality of pores are slit-shaped and whereinlength of the pores is in the range of 1 micron-5 micron, e.g., 1 μm-3μm.

Also disclosed herein are methods for transplanting the devices providedin the subject application into a patient. In certain cases, a methodfor providing a bioartificial ultrafiltration device comprising cells toa subject in need thereof includes connecting the bioartificialultrafiltration device comprising a planar scaffold comprising a matrixcomprising a population of cells and a plurality of channels adjacent tothe population of cells, wherein the channels extend from a firstsurface to a second surface of the planar scaffold; a firstsemipermeable ultrafiltration membrane disposed on the first surface ofthe planar scaffold; a first compartment adjacent to the first surfaceof the planar scaffold and in fluidic communication with the planarscaffold via the first semipermeable ultrafiltration membrane andcomprising an inlet and an outlet; a second compartment adjacent to thesecond surface of the planar scaffold and comprising an outlet, whereinthe first semipermeable ultrafiltration membrane comprises a pluralityof pores having a width in the range of 5 nm-5 micron, wherein the firstsemipermeable ultrafiltration membrane allows transport of ultrafiltratefrom the first compartment to the plurality of channels and wherein theultrafiltrate traverses from the plurality of channels into the secondcompartment, to the subject, wherein the connecting comprises connectingthe inlet of the first compartment to an artery of the subject andconnecting the outlet of the first compartment to a blood vessel of thesubject; and connecting the outlet of the second compartment to a bloodvessel or a body cavity of the subject; or connecting the outlet of thesecond compartment to an analyte analysis device.

In certain embodiments, a method for providing a bioartificialultrafiltration device comprising cells to a subject in need thereofincludes connecting the bioartificial ultrafiltration device comprisinga first planar scaffold and a second planar scaffold each comprising amatrix comprising a population of cells and a plurality of channelsadjacent the population of cells, wherein the channels extend from afirst surface to a second surface of each of the planar scaffolds; afirst semipermeable ultrafiltration membrane disposed on the firstsurface of the first and second planar scaffolds; a first compartmentadjacent to and sandwiched between the first surface of the first andsecond planar scaffolds and comprising an inlet and an outlet, whereinthe first semipermeable ultrafiltration membrane allows transport ofultrafiltrate from the first compartment to the scaffolds; a secondcompartment adjacent to the second surface of the first planar scaffoldand comprising an outlet; a third compartment adjacent to the secondsurface of the second planar scaffold and comprising an outlet, whereinthe first semipermeable ultrafiltration membrane comprises a pluralityof pores having a width in the range of 5 nm-5 micron, and wherein theultrafiltrate traverses from the plurality of channels in the scaffoldsinto the second compartment and the third compartment, to the subject,wherein the connecting comprises connecting the inlet of the firstcompartment to an artery of the subject and connecting the outlet of thefirst compartment to a blood vessel of the subject; and connecting theoutlets of the second and third compartments to a blood vessel or bodycavity of the subject; or connecting the outlets of the second and thirdcompartments to an analyte analysis device; or connecting the outlet ofthe second compartment to a blood vessel or a body cavity of the subjectand connecting the outlet of the third compartment to an analyteanalysis device; or connecting the outlet of the second compartment toan analyte analysis device and connecting the outlet of the thirdcompartment to a second vein of the subject.

In another embodiment, a method for providing a bioartificialultrafiltration device comprising cells to a subject in need thereofincludes connecting the bioartificial ultrafiltration device comprisinga planar scaffold comprising a matrix comprising a population of cellsand a plurality of channels adjacent the population of cells, whereinthe channels extend from a first surface to a second surface of theplanar scaffold; a first semipermeable ultrafiltration membrane disposedon the first surface and a second semipermeable ultrafiltration membranedisposed on the second surface of the planar scaffold; a firstcompartment comprising a first inlet and a first outlet, wherein thefirst compartment is adjacent to the first surface of the planarscaffold; a second compartment comprising a second inlet and a secondoutlet, wherein the second compartment is adjacent to the second surfaceof the planar scaffold, wherein the first inlet is configured forconnection to an artery of a subject and the first outlet is connectedto the second inlet of the second compartment, wherein the second outletof the second compartment is configured for connection to a vein of thesubject, wherein the semipermeable ultrafiltration membranes comprise aplurality of pores having a width in the range of 5 nm-5 micron, whereinthe first semipermeable ultrafiltration membrane allows transport ofultrafiltrate from the first compartment to the scaffold and the secondsemipermeable ultrafiltration membrane allows transport of theultrafiltrate from the plurality of channels in the scaffold into thesecond compartment, to the subject, wherein the connecting comprisesconnecting the first inlet to an artery of a subject; and connecting thesecond outlet to a vein of the subject.

In some embodiments, the method comprises providing insulin to thesubject and wherein the cells comprise insulin producing cells. In somecases, the insulin producing cells are derived from differentiation ofstem cells or are pancreatic cells such as, pancreatic beta cellsisolated from pancreatic islets. In some examples, the cells are isletcells, for example, the cells may be present in islets isolated frompancreas. The cells may be autologous, allogenic, or xenogenic to thesubject. In certain embodiments, connecting the bioartificial device tothe subject in need thereof results in increased viability of the cellsin the scaffold. In certain embodiments, the ultrafiltrate comprises oneor more of glucose and oxygen. In certain embodiments, the ultrafiltratecomprises one or more of glucose and oxygen and wherein the insulinproducing cells excrete insulin in response to presence of glucose inthe ultrafiltrate and wherein the plurality of channels transport theinsulin to the second compartment and/or to the third compartment. Incertain embodiments, the excreted insulin is transported to theplurality of channels in the scaffold.

In certain embodiments, the semipermeable ultrafiltration membranesreduce or prevent the passage of immune system components into thescaffold, e.g., passage of antibodies into the scaffold, passage ofcytokines (e.g., TNF-α, IFN-γ, and/or IL-1β) into the scaffold.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show silicon nanoporous membranes (SNM).

FIGS. 2A-2I show a schematic for fabrication of silicon nanoporemembranes.

FIGS. 3A-3E show in vitro viability of mouse islets under cytokineexposure.

FIG. 4 shows glucose-stimulated insulin release from mouse islets in theSNM-encapsulation chamber and in static culture.

FIG. 5 shows a transport of various molecules through slit-pore of SNMunder a pressure difference of ˜2 psi.

FIG. 6 shows a conceptual illustration of the implantable intravascularbioartificial pancreas device in the arm of a T1D patient.

FIG. 7 shows a schematic diagram of the mock-loop circuit for in vitroassessment of SNM-encapsulated islets under convective conditions.

FIG. 8 shows a schematic diagram of the pressure-driven cytokinefiltration testing system.

FIG. 9 shows a schematic diagram of the hydraulic permeability testingsystem. Air was applied through a pressure regulator into the liquidreservoir.

FIG. 10 shows a. comparison of relative solute size (λ).

FIG. 11 shows an assessment of solute distribution in the mock-loopsystem.

FIG. 12A shows an SEM image of the tilted membrane surface which depictsnanopores with 2 μm in length. FIG. 12B shows an SEM image of thecross-section of the membrane which depicts nanopores with 7 nm in widthand 300 nm in depth. FIG. 12C shows an SEM image of the membrane surfacewhich depicts micropores with 4 μm in length. FIG. 12D shows an SEMimage of the cross-section of the membrane which depicts micropores with1 μm in width.

FIGS. 13A-13C show Glucose-insulin kinetics of SNM-encapsulated isletsunder convection and diffusion without cytokine exposure.

FIGS. 14A-14C show glucose-insulin kinetics of SNM- and siliconmicropore membrane (Sμm)-encapsulated islets under convection withoutcytokine exposure.

FIGS. 15A-15C show glucose-insulin kinetics of SNM-encapsulated isletsunder convection and diffusion with cytokine exposure.

FIGS. 16A-16C show glucose-insulin kinetics of SNM- and SμM-encapsulatedislets under convection with cytokine exposure.

FIGS. 17A-17B show in-vitro viability of mouse islets.

FIG. 18 shows the rate of change in insulin secretion without cytokineexposure in Table 1.

FIG. 19 shows the rate of change in insulin secretion with cytokineexposure in Table 2.

FIGS. 20A-20D show an illustration of the process and fixtures for CellScaffold/islet chamber (IC) construction. The terms cell scaffold andislet chamber are used interchangeably.

FIG. 21 shows a zoomed-in view of the components of the bioartificialdevice.

FIGS. 22A-22B show the inlet and outlet components of the bioartificialdevice.

FIGS. 23A-23B show an illustration of the bioartificial device connectedinline to an arterial-venous graft and an ultrafiltrate catheterdelivering insulin rich ultrafiltrate to a vein.

FIGS. 24A-24C show glucose-insulin kinetics of SμM-encapsulated isletsunder convection and diffusion without cytokine exposure.

FIGS. 25A-25C show glucose-insulin kinetics of SNM- and SμM-encapsulatedunder diffusion without cytokine exposure.

FIGS. 26A-26C show glucose-insulin kinetics of SμM-encapsulated isletsunder convection and diffusion with cytokine exposure.

FIGS. 27A-27C show glucose-insulin kinetics of SNM- and SμM-encapsulatedislets under diffusion with cytokine exposure.

FIGS. 28A-28B show in-vitro viability of mouse islets.

FIG. 29 shows the rate of change in insulin secretion as depicted in thetable.

FIGS. 30A-30B show SEM images of the pore-containing regions surroundedby solid silicon regions.

FIG. 31 shows a gross image of islets and agarose mixture inside the ICin which the maximum diameter surrounding each ultrafiltrate channel is800 μm.

FIGS. 32A-32C shows in vitro testing of the intravascular bioartificialpancreas device (iBAP) with 10% or 20% islet density encapsulated with10 nm-pore size SNM.

FIGS. 33A-33C shows in vitro testing of the intravascular bioartificialpancreas device (iBAP) with 10% or 20% islet density encapsulated with40 nm-pore size SNM.

FIGS. 34A-34D shows in vivo testing of the intravascular bioartificialpancreas device (iBAP) with 5% islet density encapsulated with 10nm-pore size SNM for 3 days.

FIGS. 35A-35D shows in vivo testing of the intravascular bioartificialpancreas device (iBAP) with 10% islet density encapsulated with 10nm-pore size SNM under either diffusion or convection for 3 days.

FIGS. 36A-36B shows blood flow in the iBAP.

FIG. 37 shows daily measurement of the systematic cytokine concentrationin the pig.

FIG. 38 shows daily measurement of the systematic cytokine concentrationin the pig.

FIG. 39 shows silicon nanopore membrane (SNM) hydraulic permeability asa function of pore size.

FIG. 40 shows SEM images of uncoated (left) and PEG-coated (right)silicon surfaces at low (top) and high (bottom) magnification after 30days of blood exposure in vivo in femoral vessels of anticoagulant freerodents.

FIG. 41 shows SNM encapsulation of islets provides immunoisolation fromcytokines and retains islet viability. IL-1β, TNF-α, and IFN-γ weretested at 50 U/ml, 1000 U/ml and 1000 U/ml respectively.

FIG. 42 shows a schematic of the mock circuit loop for in vitroassessment of SNM encapsulated islets under convection.

FIG. 43 shows islet in vitro glucose-insulin kinetics data.

FIG. 44 shows an image of the prototype full-scale iBAP connected to theporcine vasculature on the day of implant.

FIGS. 45A-45C show the structural layout of the Islet Chamber. FIG. 45Ashows an isometric view of a 6 mm×6 mm×1.2 mm Islet Chamber that will beplaced within the SNM support structure cavity. FIG. 45B shows a closeup of the Islet Chamber corner with the Fluid Channel (FC) and thedirection of ultrafiltrate flow. FIG. 45C illustrates a top view of theIslet Chamber's Islet Volume (IV), Structural Volume (SV) and FC regionslabeled along with dimensions.

FIG. 46 shows cell scaffold design features to optimize.

FIGS. 47A-47B show views of a PDMS mold. FIG. 47A shows an isometricview of a 6 mm×6 mm×1.2 mm PDMS positive mold with cells/hydrogelmixture. FIG. 47B shows a close up where the PDMS posts and structuralbase are gray, the cells are green, and the hydrogel is a translucentpurple.

FIG. 48 shows Islet viability data from the in vitro experiments.

FIG. 49 shows Islet in vitro glucose-insulin kinetics data.

FIG. 50 shows Islet Chamber design features to optimize.

FIG. 51 shows the process flow used to fabricate SNM.

FIG. 52 shows complement mediated lysis of ovine erythrocytes forvarious membrane.es SNM clearly outperform conventional polymermembranes.

FIG. 53 shows rapid glucose-stimulated insulin response of SNMencapsulated islets.

FIG. 54 shows fibrinogen deposition over a 1-month period on siliconcoated substrates with various molecular coatings. PVAm and polySBMAappear suitable for long-term implantation.

FIG. 55 shows a cross-sectional view of SNM and Islet Chamber.

FIGS. 56A-56B show a schematic of a connector for connecting a graft toa device as disclosed.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

As used herein, the term “filtration” refers to a process of separatingparticulate matter from a fluid, such as air or a liquid, by passing thefluid carrier through a medium (e.g., a semipermeable membrane) thatwill not pass the particulates.

As used herein, the term “ultrafiltration” refers to subjecting a fluidto filtration, where the filtered material is very small; typically, thefluid comprises colloidal, dissolved solutes or very fine solidmaterials, and the filter is a microporous or nanoporous. The filter maybe a membrane, such as, a semi-permeable membrane. The fluid to befiltered is referred to as the “feed fluid.” In certain embodiments, thefeed fluid may be arterial blood. During ultrafiltration, the feed fluidis separated into a “permeate” or “filtrate” or “ultra-filtrate,” whichhas been filtered through the filter, and a “retentate,” which is thatpart of the feed fluid which did not get filtered through the membrane.

As used herein the terms “subject” or “patient” refers to a mammal, suchas, a primate (e.g., humans or non-human primates), a bovine, an equine,a porcine, a canine, a feline, or a rodent. In certain embodiments, thesubject or patient may be a human. In certain embodiments, the subjector patient may be pre-diabetic or may have diabetes, such as, type 1diabetes (T1D) or type 2 diabetes. The terms “subject” and “patient” areused interchangeably herein.

As used herein, the terms “treat,” “treatment,” “treating,” and thelike, refer to obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment,” as used herein,covers any treatment of a disease in a subject, particularly in a human,and includes: (a) preventing the disease from occurring in a subjectwhich may be predisposed to the disease but has not yet been diagnosedas having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; and (c) relieving the disease, e.g., causing regression ofthe disease, e.g., to completely or partially remove symptoms of thedisease.

As used herein, the terms “layer”, “film”, or “membrane” and pluralsthereof as used in the context of a device of the present disclosurerefer to an individual layer of the device that may be formed from asilicon membrane, silicon nitride, silica, atomically thin membrane suchas graphene, silicon, silicene, molybdenum disulfide (MoS₂), etc., or acombination thereof or a polymer. The “layer”, “film”, or “membrane”used to manufacture a porous layer of the present disclosure istypically porous and can be nanoporous or microporous. The phrases“nanoporous layer,” “nanopore layer,” “nanoporous membrane,” “nanoporemembrane,” “nanoporous film,” and “nanopore film” are usedinterchangeably and all refer to a polymer layer in which nanopores havebeen created. A nanoporous layer may include a frame for supporting thelayer. The phrases “microporous layer,” “micropore layer,” “microporousmembrane,” “micropore membrane,” “microporous film,” and “microporefilm” are used interchangeably and all refer to a polymer layer in whichmicropores have been created. A microporous layer may include a framefor supporting the layer.

As used herein, the term “encapsulated” as used in the context of cellsdisposed in a matrix of a scaffold as described herein. The scaffold maybe included into the devices provided herein. The cells may beencapsulated in a matrix that includes a plurality of channels adjacentthe encapsulated cells. The cells may be encapsulated in a matrix thatincludes a biocompatible polymerizable polymer.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to“channels” includes a plurality of such channels and reference to “theagarose-cell region” includes reference to one or more agarose-cellregions and equivalents thereof known to those skilled in the art, andso forth. It is further noted that the claims may be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

As summarized above, bioartificial ultrafiltration devices comprising apopulation of cells is disclosed. Also described herein are methods formaking the devices and methods of using the devices. The bioartificialdevices provided herein may be used for providing an ultrafiltrateproduced by filtration of blood across a semipermeable porous membrane.The ultrafiltrate may be provided to the population of cells and mayprovide nutrients, such as, oxygen and glucose to the cells. Theultrafiltrate may take up molecules secreted by the cells such asinsulin and other metabolites and the ultrafiltrate may be returned tothe blood. The ultrafiltrate may be enclosed in channels which aresurrounded by the cells thereby increasing the rate of exchange ofmolecules. The devices of the present disclosure can have a variety ofconfigurations and uses. The following sections provide a detaileddescription of various embodiments and uses of the device disclosedherein.

Cell Scaffold

Provided herein are scaffolds that support a population of cellsencapsulated in a matrix. In certain embodiments, the planar scaffold isthree-dimensional and includes a first surface opposite a secondsurface. The planar scaffold may be include a sheet of a solid substratein which a void has been created which void contains a matrix thatsupports the population of cells and a plurality of channels.

The solid substrate may be composed of any suitable material, such as, apolymer, such as a biocompatible polymer. Non-limiting examples ofmaterials for the solid substrate region of the scaffold can be found inU.S. Pat. No. 9,132,210, which is herein incorporated by reference inits entirety. Non-limiting examples of materials for the solid substrateregion of the scaffold: polylactic acid, polyglycolic acid, PLGApolymers, polyesters, poly(allylamines)(PAM), poly(acrylates), modifiedstyrene polymers, pluronic polyols, polyoxamers, poly(uronic acids),poly(vinylpyrrolidone), alginate, polyethylene glycol, fibrin, and poly(methyl methacrylate) and copolymers or graft copolymers of any of theabove. In certain embodiments, the solid substrate of the scaffold iscomposed of acrylate. In certain embodiments, the acrylate is in theform of an acrylate sheet.

In certain embodiments, the solid substrate may be laser-cut to create avoid. In certain embodiments, a sheet of the solid substrate islaser-cut to create a void region. In certain embodiments, a sheet ofthe solid substrate is laser-cut to create two or more void regionswhich may be cut into individual pieces. The void or cut-out may haveany shape, such as, square, or rectangular with edges that aresubstantially straight or undulating, or circular with substantiallysmooth periphery. The void may be of a size sufficient to contain aplurality of channels and a population of cells adjacent the pluralityof channels. The size of the void may be proportional to the size of thedevice. In certain cases, the solid surface may be have a thickness of0.1 mm-10 mm, such as, 0.5 mm-5 mm, or 0.5 mm-3 mm and may be in shapeof a cube or a cuboid and the surface area of the first surface and thesecond surface may 1 cm²-200 cm², e.g., 1 cm²-100 cm², 1 cm²-50 cm², 1cm²-25 cm², or 5 cm²-50 cm². The matrix may have a surface area of 1mm²-10,000 mm², e.g., 1 mm²-5000 mm², 1 mm²-1000 mm², 1 mm²-100 mm². Incertain cases the void may have a surface area of 100 cm² or less suchas, 15 cm² or less, 10 cm² or less, 5 cm² or less, 1 cm² or less, 0.5cm² or less, for example, 20 cm²-0.5 cm², 15 cm²-0.3 cm², 10 cm²-0.5cm², 5 cm²-0.5 cm², or 1 cm²-0.5 cm². The depth of the void and thematrix disposed therein is determined by the thickness of the solidsubstrate used to form the scaffold and may be in the range of 200micron to 1000 micron, such as, 200 micron-900 micron, 200 micron-800micron, 300 micron-700 micron, 200 micron-700 micron, or 300 micron-600micron. In certain embodiments, the void may have a dimension of about1-5 mm (length)×1-5 mm (width)×0.5-1 mm (depth), e.g., 1 mm×3 mm×1 mm, 2mm×3 mm×1 mm, 3 mm×3 mm×1 mm, or 4 mm×4 mm×1 mm.

In certain embodiments, the planar scaffold comprises a matrix. Incertain embodiments, the matrix comprises a plurality of channels and apopulation of cells. In certain embodiments, the matrix is formed bydisposing a plurality of elongate posts into the void where the elongateposts are oriented in a direction perpendicular to the first and secondsurfaces of the solid substrate in which the void was created. Incertain embodiments, the elongate posts may be tubes or wires, such as,polytetrafluorethlene (PFTE) coated wires. In certain embodiments, theelongate posts may be substantially cylindrical in shape. The diameterof the elongate posts may range from 25 micron-500 micron, such as, 50micron-500 micron, 50 micron-300 micron, 50 micron-200 micron, 75micron-200 micron, 75 micron-150 micron, e.g., 100 μm in diameter. Inother cases, the elongate posts may have a rectangular shape or anirregular shape. The matrix may be formed from a polymerizablebiocompatible polymer that support viability of the encapsulated cells.For example, the matrix may enable transport of molecules (e.g.,glucose, oxygen, insulin) to and from the cells. In certain embodiments,the matrix comprises a polylactic acid, polyglycolic acid, polyethyleneglycol (PEG), poly(lactic-co-glycolic acid) (PLGA) polymer, alginate,alginate derivative, gelatin, collagen, fibrin, agarose, hyaluronicacid, hydrogel, matrigel, natural polysaccharide, syntheticpolysaccharide, polyamino acid, polyester, polyanhydride,polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), modifiedstyrene polymer, pluronic polyol, polyoxamer, poly(uronic acid), orpoly(vinylpyrrolidone) polymer. In certain embodiments, the matrix isincludes an agarose polymer, an extracellular matrix, hydrogel,matrigel, or a mixture thereof. In certain embodiments, the matrixfurther includes a population of cells. In certain embodiments, thematrix is formed by disposing a composition comprising an unpolymerizedpolymer and a population of cells as disclosed herein into the voidregion of the solid substrate of the scaffold, which void regionsinclude the elongate posts. The composition is then polymerized and theelongate posts removed to provide a matrix that includes the populationof cells and a plurality of channels created by removal of the wires. Incertain embodiments, the plurality channels are cylindrical shapedchannels. In certain embodiments, the channels are rectangular,cylindrical, or square shaped. In certain embodiments, the channels areat least 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, or 150 μm in diameter. In certainembodiments, the channels 30-200 μm or 50-150 μm in diameter. In certainembodiments, the channels extend from a first surface to a secondsurface of the planar scaffold. The matrix may include two or morechannels, such as, 3-25,000, 5-10,000, 10-10,000, 30-10,000, 50-10,000,100-10,000, 1000-10,000, 100-3000, 3-100, 3-70, 3-50, 3-25, 3-20, 5-100,6-75, 7-50, or 8-20 channels where the channels are adjacent apopulation of cells. The matrix is configured such that a channel isseparated by a distance less than 500 μm from a cell in order tofacilitate efficient exchange of molecules between the ultrafiltrate inthe channel and the cell.

In certain embodiments, the scaffold includes a plurality of channelswhere at least one of the channels is surrounded by a hexagonalarrangement of the cells. In certain embodiments, at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least ten, at least eleven, at leasttwelve, at least thirteen, at least fourteen, at least fifteen, at leastsixteen, at least seventeen, at least eighteen, at least nineteen, or atleast twenty channels are surrounded by a hexagonal arrangement of thecells. In certain embodiments, the cells are adjacent at least one ofthe plurality of channels such that the cells are separated from thechannel by a distance less than 500 micron, such as, 400 micron, 300micron, or 200 micron. The presence of cells adjacent to the channelsfacilitates diffusion of molecules 500 micron from at least one channel.In certain embodiments, the matrix includes a configuration of a channelsurrounded by a hexagonal cluster of cells. In certain embodiments, thescaffold may include a cell density of at least 5% by volume, 10% (5,700cell equivalents/cm²), 20% (11,400 cell equivalents/cm²) or more. Thenumber of cells in the matrix of the bioartificial device may vary andmay be determined empirically. In some cases, the scaffold may includeat least 10³ cells, 10⁵ cells, 10⁶ cells, 10¹⁰ cells, such as 10³-10¹⁰cells, 10⁵-10⁸ cells, 10³-10⁶ cells, or 10⁵-10⁶ cells. In certainembodiments, the matrix includes a plurality of channels surrounded by apopulation of cells, where the diameter of the channel-cell region is100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm,or 1000 μm, e.g., in the range of 800 μm-1000 μm in diameter. In certainembodiments, the scaffold includes eight 800 μm channel-cell regionswith eight 100 μm diameter cylindrical channels. The scaffold thusincludes a matrix in which the cells are encapsulated followingpolymerization of the composition containing the mixture ofunpolymerized polymer and cells.

In certain cases, the matrix supported in a planar scaffold may beconfigured as depicted in FIGS. 45A-C. FIG. 45A shows an isometric viewof a 6 mm×6 mm×1.2 mm matrix that will be placed within the planarscaffold cavity. FIG. 45B shows a close up of the matrix corner with aplurality of fluid channels (FC) and the direction of ultrafiltrateflow. FIG. 45C illustrates a top view of the Islet Chamber's IsletVolume (IV), Structural Volume (SV) and FC regions labeled along withdimensions.

In certain cases, the matrix of insulin secreting cells (referred to asislet cells) may be configured according to the arrangement depicted inFIG. 47 with dimensions as listed in FIG. 46. For example, the insulinsecreting cells may be encapsulated in a matrix having a thickness of500-2000 micron and the matrix may include a defined configuration of aperiodic arrangement of channels for the flow of ultrafiltrate, whereinthe channels may traverse the thickness of the matrix and may have awidth of 10-100 micron.

Populations of cells that can be included in the devices describedherein include but are not limited to, bone marrow cells; mesenchymalstem cells, stromal cells, pluripotent stem cells (e.g., inducedpluripotent stem cells or embryonic stem cells), blood vessel cells,precursor cells derived from adipose tissue, bone marrow derivedprogenitor cells, intestinal cells, islets, Sertoli cells, beta cells,progenitors of islets, progenitors of beta cells, peripheral bloodprogenitor cells, stem cells isolated from adult tissue, retinalprogenitor cells, cardiac progenitor cells, osteoprogenitor cells,neuronal progenitor cells, and genetically transformed cells, or acombination thereof. The population of cells may be from the subject(autologous cells), from another donor (allogeneic cells) or from otherspecies (xenogeneic cells). The cells can be introduced into thescaffold and the scaffold may be immediately (within a day) implantedinto a subject or the cells may cultured for longer period, e.g. greaterthan one day, to allow for cell proliferation prior to implantation.

In certain embodiments, the populations of cells in the matrix are stemcells. In certain embodiments, the population of cells in the matrix arepancreatic progenitor cells. In certain embodiments, the population ofcells in the matrix are pancreatic cells isolated from islets ofpancreas. In certain embodiments, the population of cells in the matrixare islets isolated from pancreas. In certain embodiments, thepopulation of cells in the matrix may be in the form of a piece oftissue, such as, islet of Langerhans, which may have been isolated fromthe subject receiving the device or from another subject.

In certain embodiments, the devices disclosed herein may be used totreat a person having diabetes, such as, type 1 diabetes. The device mayinclude pancreatic islet cells or may include stem cells that arecapable of differentiating into insulin producing pancreatic cells. Incertain embodiments, pluripotent stem cells (PSCs) may be differentiatedinto insulin producing pancreatic cells inside the device and then thebioartificial device containing the differentiated insulin producingpancreatic cells is placed in the subject (e.g., in the omentum,adjacent to pancreas or liver, adjacent to kidney, lung, or heart, orsubdermally, e.g., in arm or abdomen). In some case, the device mayinclude PSCs and the device may be implanted adjacent the pancreas orliver of the subject.

Bioartificial Ultrafiltration Device

In certain embodiments, the bioartificial device may include a planarscaffold described herein, e.g., a planar scaffold that includes amatrix comprising a population of cells and a plurality of channelsadjacent to the population of cells, wherein the channels extend from afirst surface to a second surface of the planar scaffold; a firstsemipermeable ultrafiltration membrane disposed on the first surface ofthe planar scaffold; a first compartment adjacent to the first surfaceof the planar scaffold and in fluidic communication with the planarscaffold only via the first semipermeable ultrafiltration membrane andcomprising an inlet and an outlet; a second compartment adjacent to thesecond surface of the planar scaffold and comprising an outlet, whereinthe first semipermeable ultrafiltration membrane comprises a pluralityof pores having a width in the range of 5 nm-5 micron, wherein the firstsemipermeable ultrafiltration membrane allows transport of ultrafiltratefrom the first compartment to the plurality of channels and wherein theultrafiltrate traverses from the plurality of channels into the secondcompartment.

In certain embodiments, the bioartificial device may include two planarscaffolds which sandwich a compartment containing arterial blood. Forexample, the device may include a first planar scaffold and a secondplanar scaffold each comprising a matrix comprising a population ofcells and a plurality of channels adjacent the population of cells,wherein the channels extend from a first surface to a second surface ofeach of the planar scaffolds; a first semipermeable ultrafiltrationmembrane disposed on the first surface of the first and second planarscaffolds; a first compartment adjacent to and sandwiched between thefirst surface of the first and second planar scaffolds and comprising aninlet and an outlet, wherein the first semipermeable ultrafiltrationmembrane allows transport of ultrafiltrate from the first compartment tothe scaffolds; a second compartment adjacent to the second surface ofthe first planar scaffold and comprising an outlet; a third compartmentadjacent to the second surface of the second planar scaffold andcomprising an outlet, wherein the first semipermeable ultrafiltrationmembrane comprises a plurality of pores having a width in the range of 5nm-5 micron, and wherein the ultrafiltrate traverses from the pluralityof channels in the scaffolds into the second compartment and the thirdcompartment.

Aspects of the present disclosure include a bioartificial device thatincludes a planar scaffold comprising a matrix comprising a populationof cells and a plurality of channels adjacent the population of cells,where the channels extend from a first surface to a second surface ofthe planar scaffold; a first semipermeable ultrafiltration membranedisposed on the first surface and a second semipermeable ultrafiltrationmembrane disposed on the second surface of the planar scaffold; a firstcompartment comprising a first inlet and a first outlet, wherein thefirst compartment is adjacent to the first surface of the planarscaffold; a second compartment comprising a second inlet and a secondoutlet, wherein the second compartment is adjacent to the second surfaceof the planar scaffold, wherein the first inlet is configured forconnection to an artery of a subject and the first outlet is connectedto the second inlet of the second compartment, where the second outletof the second compartment is configured for connection to a vein of thesubject, where the semipermeable ultrafiltration membranes comprise aplurality of pores having a width in the range of 5 nm-5 micron, wherethe semipermeable ultrafiltration membrane allows transport ofultrafiltrate filtered from the arterial blood in the first compartmentto the scaffold and transport of the ultrafiltrate from the plurality ofchannels in the scaffold into the second compartment. In certain cases,the first outlet may include a means for reducing the rate of flow ofblood to the second compartment. In some case, the means may include apressure reduction manifold. In some cases, the means for reducing therate of flow of blood to the second compartment may include a pressurereduction channel. In some cases, the rate of flow of blood through thesecond compartment may be controlled by directing the blood through achannel having a reduced width compared to the width of the firstcompartment. In some case, the rate of flow of blood through the secondcompartment may be controlled by sizing the second compartment to have areduced width compared to the width of the first compartment.

In some cases, the first compartment into which the blood is introducedinto the device may have a dimension suitable for facilitatingultrafiltration of the blood. For example, the first compartment mayhave a height of 100 micron-6 mm, e.g., 500 micron-4 mm, 1 mm-3 mm, or 2mm-3 mm.

In certain embodiments, the bioartificial device is dimensioned to fitin a body cavity of a subject. The device may be rectangular orcylindrical in shape. In certain case, the device may have a surfacearea of 50 cm² or less, such as 10-30 cm², 10-25 cm², 15-25 cm², 20-25cm², 15-30 cm². In certain cases, the device may be rectangular and havea length of 3 cm-10 cm, a width of 1 cm-6 cm, and a height of 0.3 cm-2cm, such as dimension (length×width×height) of 3 cm×1 cm×0.5 cm to 6cm×4 cm×1 cm, e.g., 3 cm×1 cm×0.5 cm, 5 cm×2 cm×1 cm, or 6 cm×4 cm×1 cm.

As noted herein, the devices disclosed herein may maintain thetransplanted cells in a functional and viable state for at least 1 monthand up to a period of at least 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 3years, 5 years, 10 years, or up to 50 years, or longer, such as, 1month-50 years, 1 year-25 years, 5 years-50 years, 5 years-25 years, 10years-50 years, or 15 years-25 years.

In certain embodiments, the devices disclosed herein may be enclosed ina housing made from an inert material that does not degrade or foul whenplaced in a subject. Any material approved for medical devices placed ina subject may be utilized including but not limited to medical gradeplastic, inert metals, such as, titatnium, stainless steel, etc.

In certain embodiments, the bioartificial device comprises more than onesemipermeable ultrafiltration membrane. In certain embodiments, thesemipermeable ultrafiltration membrane is disposed on the first surfaceand the second surface of the planar scaffold. The semipermeableultrafiltration membrane disposed on a first surface of the scaffold maybe the same as the semipermeable ultrafiltration membrane disposed onthe second surface of the scaffold or may be different. For example, thesemipermeable ultrafiltration membrane adjacent to a compartmentcontaining arterial blood may have smaller pores than the semipermeableultrafiltration membrane adjacent a compartment containing theultrafiltrate flowing through the channels in the matrix. In some cases,the semipermeable ultrafiltration membrane adjacent to a compartmentcontaining arterial blood may have larger pores than the semipermeableultrafiltration membrane adjacent a compartment containing theultrafiltrate flowing through the channels in the matrix. In certainembodiments, the semipermeable membrane allows for filtration of anultrafiltrate from the compartment containing arterial blood whichultrafiltrate is transported into the plurality of channels in thescaffold. The plurality of channels are adjacent the cells whichprovides for efficient exchange of molecules in the ultrafiltrate in thechannels with the molecules released by the cells. These moleculesdiffuse in a concentration dependent manner between the lumen of thechannels and the matrix surrounding the cells. For example, molecules,such as oxygen, glucose, lipids, vitamins, and minerals diffuse from thelumen of the channels into the matrix to the cells and moleculessecreted by the cells such as urea, carbon dioxide, insulin aretransported into the lumen of the channels. It is understood that insome embodiments, the some diffusion and exchange of molecules in theultrafiltrate may occur outside of the channels, such as, with theultrafiltrate that does not enter the channels and permeates through thematrix.

In certain embodiments, the semipermeable ultrafiltration membranecomprises a plurality of pores having a width in the range of 5 nm-5micron. In some embodiments, the present disclosure provides a membranecomprising fabricated pores of defined dimensions and structure, anddensity. In certain embodiments, one or more surface of the membrane maybe treated to limit protein adsorption. Such a treatment may includetreatments that alter or confer surface charge, surface free energy, ortreatments that promote adhesion of specific cell types. In certainembodiments, at least one pore of the membrane comprises any combinationof a surface treatment. Surface treatments function to effectrestriction of size and electrostatic charge of solutes that may bepassed through such pores. Examples of surface treatments can be found,for example, in U.S. Patent Application Publication No. 20090131858,which is hereby incorporated by reference in its entirety.

In certain embodiments, the semipermeable ultrafiltration membrane isconfigured for filtration of biological fluids. In certain embodiments,the membrane comprises a plurality of nanopores, where the shapes andsizes of the pores are controlled. In certain embodiments, the membranecomprises a plurality of pores. In certain embodiments, the plurality ofpores may be micropores and have a width in the range of 0.1 μm-5 μm,e.g., 0.1 μm-3 μm, 0.1 μm-0.5 μm, 0.5 μm-1 μm, 1 μm-1.5 μm, 1.5 μm-2 μm,0.1 μm-1 μm, 0.1 μm-0.8 μm, 0.2 μm-0.7 μm, 0.2 μm-0.6 μm, 0.2 μm-0.5 μm.In certain embodiments, the plurality of pores may be nanopores and mayhave a width of 1 nm-500 nm, e.g., 1 nm-90 nm, 2 nm-50 nm, 3 nm-40 nm, 4nm-50 nm, 4 nm-40 nm, 5 nm-50 nm, 5 nm-20 nm, 4 nm-20 nm, 7 nm-100 nm,12 nm-20 nm, or 5 nm-10 nm. In certain embodiments, the plurality ofpores are slit shaped and have a width as listed herein and have alength in the range of 1 μm-10 μm, e.g., 2 μm-3 μm, 3 μm-4 μm, 4 μm-5μm, 5 μm-6 μm, 6 μm-7 μm, 7 μm-8 μm, 8 μm-9 μm, or 9 μm-10 μm. Incertain cases, the rectangular pores have a depth of 100-1000 nm, awidth of 3 nm-50 nm and a length of 1 micron-5 micron, e.g., awidth×length×depth of 5 nm-50 nm×1 micron-2 micron×200 nm-500 nm.

In certain embodiments, the devices of the present disclosure includesemipermeable ultrafiltration membranes having a dimension(length×width) of 6 mm×6 mm, 5 mm×5 mm, 7 mm×7 mm, 8 mm×8 mm, 9 mm×9 mm,10 mm×10 mm, 10 cm×10 cm, e.g., 10 mm×10 mm to 10 cm×10 cm. In someembodiments, the semipermeable ultrafiltration membrane may berectangular.

In certain embodiments, the semipermeable ultrafiltration membrane has asurface area in the range of 0.5-100 cm², e.g., 30-100 cm², 10-30 cm²,15-30 cm², 15-20 cm², 20-25 cm², 25-30 cm², 0.5-10 cm², 0.75-5 cm²,0.75-3 cm², or 0.75-2 cm².

In certain embodiments, the devices disclosed herein may besubstantially planar and may be dimensioned to have a surface arearanging from 20-100 cm² (on each planar side) and a thickness of 1 cm-3cm. In certain embodiments, the devices disclosed herein may have avolume of up to 500 cm³, such as, 50-500 cm³, 100-500 cm³, 100-300 cm³,100-150 cm³. In certain cases, the device may include a semipermeablemembrane having a surface of 5-75 cm², e.g., 5-50 cm², 10-30 cm², or15-30 cm². The size of pores in the membrane may be 10 nm-100 nm inwidth, such as, 10 nm-20 nm.

The semipermeable ultrafiltration membranes of the present disclosureinclude any membrane material suitable for use in filtering biologicalfluids, wherein the membranes are structurally capable of supportingformation of pores. Examples of suitable membrane materials are known inthe art and are described herein.

In certain embodiments, the membrane material is synthetic, biological,and/or biocompatible (e.g., for use outside or inside the body).Materials include, but are not limited to, silicon, which isbiocompatible, coated silicon materials, polysilicon, silicon carbide,ultrananocrystalline diamond, diamond-like carbond (DLC), silicondioxide, PMMA, SU-8, and PTFE. Other possible materials include metals(for example, titanium), ceramics (for example, silica or siliconnitride), and polymers (such as polytetrafluorethylene,polymethylmethacrylate, polystyrenes and silicones). Materials formembranes can be found in, for example U.S. Patent ApplicationPublication No. 20090131858, which is hereby incorporated by referencein its entirety.

A semipermeable ultrafiltration membrane of the present disclosurecomprises a plurality of pores, where pore shapes include linear,square, rectangular (slit-shaped), circular, ovoid, elliptical, or othershapes. As used herein, width of a pore refers to the diameter where thepore is circular, ovoid or elliptical. In certain embodiments, themembrane comprises pores comprising a single shape or any combination ofshapes. In certain embodiments, the sizes of pores are highly uniform.In certain embodiments, the pores are micromachined such that there isless than 20% size variability, less than 10% size variability, or lessthan 5% size variability between the dimensions of the slit-shapedpores. In certain embodiments, factors that determine appropriate poresize and shape include a balance between hydraulic permeability andsolute permselectivity. In certain embodiments, the plurality of poresare slit-shaped pores which provide for optimum flux efficiency enablingefficient transport of molecules across the membrane. In certainembodiments, the membrane comprises slit-shaped nanopores. In certainembodiments, the semipermeable ultrafiltration membrane hasapproximately 10³-10⁸ rectangular slit-shaped nanopores (e.g., 10⁴-10⁸,or 10⁵-10⁷) for example on a membrane surface area of 1 cm², 0.5 cm², or0.4 cm². In certain embodiments, the number of slit-shaped nanopores onthe semipermeable ultrafiltration membrane is sufficient to allow themembrane to generate physiologically sufficient ultrafiltration volumeat capillary perfusion pressure. In certain embodiments, the porosity ofthe semipermeable ultrafiltration membrane is approximately 1%-50%,e.g., 10%-50%, 20%-50%, or 20%-75%, etc.).

In certain embodiments, the present disclosure provides a series ofmembranes comprising sparse arrays of monodisperse slit-shaped pores,manufactured, for example, using silicon bulk and surface micromachiningtechniques (see e.g., Fissell W H, et al., JAm Soc Nephrol2002; 13:602A; incorporated herein by reference in its entirety). In certainembodiments, the semipermeable ultrafiltration membrane is prototypedusing microelectromechanical systems (MEMS) technology. In certainembodiments, the process uses the growth of a thin SiO₂ (oxide) layer on400 μm-thick double side polished (DSP) silicon wafers followed by a lowpressure chemical vapor deposition (LPCVD) of polysilicon (˜500 nm). Incertain embodiments the wafers are then specifically patterned, dryoxidized, wet etched, deposited with a second polysilicon layer, andfinally blanket-etched until 400 nm of polysilicon remains and theunderlying vertical oxide layer is exposed. In certain embodiments, thevertical sacrificial oxide layer defines the critical nanoscale poresize of the membranes. In certain embodiments, the low temperature oxide(LTO) (˜1 μm) is deposited onto polysilicon of the wafers to serve asthe hard mask for membrane protection. In certain embodiments, deepreactive ion etching (DRIE) removes the backside of each window untilmembranes were disclosed. In certain embodiments, the sacrificial oxideis etched away in 49% hydrofluoric acid (HF) during the final step ofthe fabrication process to leave behind open nanoscale slit pores. Incertain embodiments, the wafers are subsequently cut into 1 cm×1 cmchips with an effective area of 6 mm²×6 mm² containing 1500 windowseach, with a total of 10⁶ pores per membrane. In certain embodiments,each rectangular pore is 7 nm in width, 300 nm in depth, and 2 μm inlength. In certain embodiments, silicon micropore membrane (SμM) isfabricated to produce wafer-scale arrays of 500 nm deep by 4 μm longrectangular slit pores with 1000 nm-wide slit width using similarprocess. In certain embodiments, the wafers are diced to form 1 cm×1 cmchips with an effective area of 6×6 mm² containing 1500 windows each,with a total of 3.12×10⁶ pores per membrane. In certain embodiments, allmembranes may be cleaned using a conventional “piranha” clean procedure,which involve a 20 min-immersion in 3:1 sulfuric acid (H₂SO₄)/hydrogenperoxide (H₂O₂) mixture, followed by thorough rinses in deionized (DI)water.

In certain embodiments, the semipermeable ultrafiltration membrane ismodified with PEG. Techniques for modification with PEG is well-known inthe art, for example, in Papra et al. 2001 (Papra, A, et al., Langmuir2001, 17 (5), 1457-1460.) In certain embodiments, the semipermeableultrafiltration membrane is covalently modified with PEG. In certainembodiments, the surface modification with PEG prevents or minimizesprotein fouling on the membrane surface. In certain embodiments, thetechnique used for PEG attachment involves a single reaction step whichcovalently couples silicon surface silanol group (Si—OH) to a chain ofPEG polymer through a trimethoxysilane group forming a Si—O—Si—PEGsequence. In such embodiments, semipermeable ultrafiltration membraneswere immersed in a solution of 3 mM2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEG-silane) (Gelest:SIM6492.7) in toluene for 2 hrs at 70° C. In certain embodiments, aseries of extensive washing steps involving toluene, ethanol, and DIwater were used to rinse away unbounded PEG residue.

In certain embodiments, the compartments of the disclosed devices are ofany appropriate shape and configuration to be compatible with thesemipermeable ultrafiltration membranes and scaffold(s) included in thedevices.

In certain embodiments, a device of the present disclosure may be asdepicted in FIGS. 21 and 22A-22B. The device depicted in FIG. 21includes a first compartment with an inlet that is connected to a tubing12 for connection to an artery and an outlet connected to a tubing 13for connection to a vein or an artery of a subject. The firstcompartment is shaped substantially as a cube or a cuboid and is closedon all sides other than an opening of the inlet, an opening of theoutlet and an opening on a first side. This open first side is closedusing a semipermeable membrane 10′ as described herein. Thesemipermeable membrane allows for limited fluid connection between thefirst compartment and the scaffold 11. The first compartment is enclosedon all other sides with an impermeable biocompatible material. Thesemipermeable membrane 10′ may be disposed on the open first side of thefirst compartment using a flexible compressible material such as arubber support or a gasket. The scaffold with the plurality of channelsand a population of cells may be disposed on the semipermeable membrane10′. A second semipermeable membrane 10 may be supported by a frame of aflexible compressible material and may be disposed on the scaffold 11. Asecond compartment that is shaped substantially as a cube or a cuboidand is closed on all sides other than an outlet (for connection to avein or body cavity of the subject or an analyte detection device, e.g.,for measuring concentration of glucose and/or insulin in theultrafiltrate) and a first open surface that is adjacent thesemipermeable membrane 10. FIG. 22A depicts an assembled device whichincludes tubing 12 that is configured for connection to an artery andtubing 13 that is configured for connection to an artery or a vein of asubject and an ultrafiltrate outlet for connection to a second vein or abody cavity of the subject or to an analyte analysis device. Asexplained herein, the first compartment is in fluid communication withthe first surface of the planar scaffold 11. In certain embodiments, thesecond compartment 20 is in fluid communication with the second surfaceof the planar scaffold 11. In certain embodiments, the first compartmentis in fluid communication with the first surface of the planar scaffold11 only by means of the pores within the membrane 10′. In certainembodiments, the second compartment is in fluid communication with thesecond surface of the planar scaffold 11 only by means of the poreswithin the membrane 10. In certain cases, the second semipermeablemembrane 10 may include pores that are larger in size than the pores inthe first semipermeable membrane 10′. In certain cases, the secondsemipermeable membrane 10 may include pores that are smaller in sizethan the pores in the first semipermeable membrane 10′. In certaincases, the device may not include the second semipermeable membrane 10.In these embodiments, the ultrafiltrate may flow from the plurality ofchannels in the scaffold to the second compartment.

In certain embodiments, the device may include two scaffolds to providean increased amount of ultrafiltrate. For example, such a device may beconfigured as depicted in FIGS. 23A and 23B and 36A-36B. The device 25includes a tubing 12 configured for supplying blood to first compartment26 via an inlet in the first compartment. The tubing may be connected toan artery. The device may include a tubing 13 for returning the blood tothe subject and may be connected to an outlet of the first compartment26 and to an artery or a vein of the subject. The device 25 alsoincludes a tubing 9 for transporting ultrafiltrate from an ultrafiltrateoutlet connected to ultrafiltrate chambers 30 a and 30 b. FIG. 23B showsa perpendicular cross section of the device of FIG. 23A. FIG. 23Bdepicts the blood channel (first compartment 26) that includes an inletfor entry of arterial blood and an outlet for exit of the blood intoback to the subject. The first compartment 26 is sandwiched between twoscaffolds 11 a and 11 b across which ultrafiltrate moves into theultrafiltrate chambers (second compartment 30 a and third compartment 30b). Also depicted is a first membrane (10 a) adjacent the firstcompartment 26 and a second membrane (10 b) adjacent the ultrafiltratecompartments (second compartment 30 a and third compartment 30 b). Theultrafiltrate compartments (second compartment 30 a and thirdcompartment 30 b) are connected to channels 31 a and 31 b which mergeinto a single channel 33 and is configured for connection to conduit 9for connecting to a vein of the subject. As noted herein, in someembodiments, the device may not include the second membranes 10 b. Insome embodiments, the second membrane 10 b may have pores that arelarger in width than the pores in the first membrane 10 a. In someembodiments, the second membrane 10 b may have pores that are smaller inwidth than the pores in the first membrane 10 a.

Another exemplary device is depicted in FIG. 6. The device 40 includes afirst compartment 49 with an inlet 41 for connection to an artery of asubject and an outlet 42 connected to a second compartment 50 via itsinlet 43. The second compartment 50 includes an outlet 44 for returningthe blood to a vein of the subject. In this embodiment, theultrafiltrate returns to the subject via the second compartment 50 aftertraversing through the second membrane 48 a. The ultrafiltrate isproduced by filtration of arterial blood across the first membrane 48 b.The ultrafiltrate carries metabolites exchanged with the cells in thescaffold 45 into the second compartment 50. As noted herein, in someembodiments, the device may not include the second membrane 48 a. Insome embodiments, the second membrane 48 a may have pores that arelarger in width than the pores in the first membrane 48 a.

In certain aspects, the scaffold may also be referred to as an isletchamber (IC) or a cell scaffold and may be formed as depicted in FIGS.20A-20D. FIG. 20A depicts a substantially planar solid substrate 15disposed on a supporting substrate 14. The solid substrate 15 includes avoid 2 that has been formed by removal of a portion of the solidsubstrate 15. The supporting substrate includes a plurality of holes orindents (3) to hold elongate posts (e.g., wires or tubes) in asubstantially straight orientation (perpendicular to the first andsecond surfaces of the solid substrate 15). FIG. 20B depicts wires 4inserted into the holes 3 of the supporting layer 15. The periphery ofthe void is marked by the border 1. FIG. 20C illustrates the void filledwith a matrix 5 that is polymerized and/or solidified (e.g., at a lowertemperature). As discussed in the foregoing sections, the matrix alsoincludes a population of cells. After the matrix is set (e.g., viapolymerization), the wires 4 are removed to reveal a plurality ofchannels 6 which extend from a first surface 7 to a second surface 8 ofthe scaffold 11 (FIG. 20D). In certain aspects, the plurality ofchannels may have a rectangular periphery, such as depicted in FIGS.47A-47C. FIGS. 47A-47C show a schematic of a region of the scaffold ofthe present disclosure which includes a plurality of rectangular shapedchannels adjacent a population of cells.

FIGS. 55A and 55B show a schematic of a connector for connecting a graftto a device as disclosed. The capsule-graft connector will affix thebioartificial device to vascular grafts and provide a blood flow pathbetween the device and the vascular grafts on both the arterial andvenous ends. FIG. 55A illustrates a device-graft connector design andFIG. 55B is a cross-sectional depiction of this design. Part 1 affixesthe connector to the device's blood port (inlet). Part 2 provides theblood flow path from the vascular graft to the device and may possess atapered lumen that gradually transitions blood from the larger diametervascular graft to the smaller diameter blood port (inlet, not shown) ofthe device. Part 3 affixes the vascular graft (Part 4) to Part 2 bycompressing the vascular graft to Part 2. Part 4 (vascular graft)provides the blood path between the device-graft connector and eitherthe arterial or venous vessel anastomosis site (not shown).

In certain embodiments, the devices provided herein provide formation ofultrafiltrate by convection rather than predominantly by diffusionthereby increasing the efficiency of ultrafiltrate formation and/or flowof the ultrafiltrate back to the subject.

Methods

The bioartificial devices of the present disclosure may be used fortransporting nutrients to a planar scaffold comprising the population ofcells in the device and release ultrafiltrate fluid containing moleculessecreted by the cells to a subject. In certain embodiments, the cellsare progenitor cells. In certain embodiments, the cells are pancreaticcells, such as, insulin producing cells isolated from pancreatic isletsor derived by differentiation of stem cells (e.g., induced pluripotentstem cells). In certain embodiments, the cells excrete insulin. Incertain embodiments, the cells release insulin into the ultrafiltratefluid.

In certain embodiments, the bioartificial device of the presentdisclosure having semipermeable ultrafiltration membranes with pores areused for protecting cells encapsulated in the scaffolds described hereinfrom an attack by the immune system of the subject. In certainembodiments, the bioartificial device of the present disclosure canreduce passage of immune system components such as cells, immunefactors, such as, antibodies and cytokines. In certain embodiments, thebioartificial device of the present disclosure can reduce passage ofimmune factors. In certain embodiments, the bioartificial device of thepresent disclosure can reduce passage of cytokines. In certainembodiments, the bioartificial device of the present disclosure canreduce passage of TNF-α, IFN-γ, and/or IL-1β while permitting transportof nutrients from the blood of the subject to the cells in the device.In certain embodiments, the bioartificial device of the presentdisclosure can reduce passage of components of the immune system (e.g.,immune cells, antibodies, cytokines, such as, TNF-α, IFN-γ, and/orIL-1β) by at least 50% (e.g., 60%-80%). In certain embodiments, thebioartificial device of the present disclosure having semipermeableultrafiltration membranes with nanopores (e.g. slit-shaped nanoporemembranes with a PEG surface coating) are used for exchange of nutrientsand small molecules to the population of cells. In certain embodiments,the bioartificial device may limit the diffusion of cytokines andimmunoglobulins through the semipermeable ultrafiltration membrane.

In certain embodiments, the bioartificial device of the presentdisclosure comprising a population of pancreatic islet cells in thematrix increase pancreatic islet cell viability within the matrix.

The bioartificial device of the present disclosure are sized to house aneffective number for cells within the bioartificial device for treatmentof a subject in need thereof. For example, the subject may be sufferingfrom a condition caused by lack of functional cells, e.g., whereinmolecules typically secreted by functional cells are not secreted or aresecreted at a level resulting in the condition. Providing functionalcells within the bioartificial device of the present disclosure couldalleviate the condition. Exemplary conditions include type 1 diabetes,Parkinson's disease, muscular dystrophy and the like.

The device may be transplanted into any suitable location in the body,such as, subcutaneously, intraperitoneally, or in the brain, spinalcord, pancreas, liver, uterus, skin, bladder, kidney, muscle and thelike. The site of implantation may be selected based on thediseased/injured tissue that requires treatment. For treatment of adisease such as diabetes mellitus (DM), the device may be placed in aclinically convenient site such as the subcutaneous space or theomentum. The device may be connected to the vascular system of thesubject as described herein. In some case, the device may be connectedinline to a vascular graft. In some cases, the device may be connectedto the subject to supply the ultrafiltrate to an artery, a vein, a bodycavity (e.g., peritoneal cavity), or a combination thereof, of thesubject. In some cases, the device may be connected to a catheter tosupply the ultrafiltrate to a vein to which the catheter is connected.

In certain cases, the device may be connected to an artery of thesubject and may supply the ultrafiltrate back to the same artery (e.g.,at a connection downstream to the site at which the artery was connectedto the device). In certain cases, the device may be connected to anartery of the subject and may supply the ultrafiltrate to a vein of thesubject. In certain cases, the device may be connected to an artery ofthe subject and may supply the ultrafiltrate to a body cavity of thesubject. In some embodiments, the device may include a sampling port forsampling the ultrafiltrate, for example, using an analyte analysisdevice for measuring concentration of insulin and/or glucose in theultrafiltrate.

In certain embodiments, the bioartificial device disclosed herein may beused to treat a person having diabetes, such as, type 1 diabetes. Thedevice may include pancreatic islet cells or may include stem cells thatare capable of differentiating into pancreatic islet cells. In certainembodiments, pluripotent stem cells (PSCs) may be differentiated intopancreatic islet cells inside the device. In some case, the device mayinclude PSCs and the device may be implanted adjacent the pancreas orliver of the subject.

As noted herein, the devices disclosed herein may maintain thetransplanted cells in a functional and viable state for at least 1 monthand up to a period of at least 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, or up to ayear or longer. In some embodiments, the present device provideimmunoisolation while supporting supply of essential components toenable islets, beta cells, and other insulin producing cells to remainviable and functional for treatment of diabetes.

The methods and devices disclosed herein can be used for both humanclinical and veterinary applications. Thus, the subject or patient towhom the bioartificial device is administered can be a human or, in thecase of veterinary applications, can be a laboratory, agricultural,domestic, or wild animal. The subject devices and methods can be appliedto animals including, but not limited to, humans, laboratory animalssuch as monkeys and chimpanzees, domestic animals such as dogs and cats,agricultural animals such as cows, horses, pigs, sheep, goats, and wildanimals in captivity such as bears, pandas, lions, tigers, leopards,elephants, zebras, giraffes, gorillas, dolphins, and whales.

In operation, blood is directed from a patient's vasculature (i.e.artery) into the inlet of the first compartment of the bioartificialdevice. Blood flows through the first compartment of the bioartificialdevice, and nutrients and small molecules from the blood are passedthrough the semipermeable ultrafiltration membrane, while largemolecules, such as immunoglobulins and cytokines within the blood areprevented from coming in contact with the cells in the device. Nutrientsand small molecules include, but are not limited to glucose, oxygen, andinsulin. The small molecules and nutrients that pass through thesemipermeable ultrafiltration membrane are filtered to form anultrafiltrate which contacts the matrix of the device comprising thepopulation of cells. In certain embodiments, the population of cellsrelease insulin into the ultrafiltrate. The ultrafiltrate then passesthrough the ultrafiltrate channels of the matrix, which then passesthrough a second semipermeable ultrafiltration membrane. Optionally, theoutlet of the second compartment can be configured to connect to acatheter. In certain embodiments, the catheter connects to the secondvein.

The disclosed devices provide a high rate of ultrafiltration creatingultrafiltrate at the rate of 1-15 ml/min at physiological rate of bloodflow.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., room temperature (RT); base pairs (bp); kilobases (kb); picoliters(pl); seconds (s or sec); minutes (m or min); hours (h or hr); days (d);weeks (wk or wks); nanoliters (nl); microliters (ul); milliliters (ml);liters (L); nanograms (ng); micrograms (ug); milligrams (mg); grams((g), in the context of mass); kilograms (kg); equivalents of the forceof gravity ((g), in the context of centrifugation); nanomolar (nM);micromolar (uM), millimolar (mM); molar (M); amino acids (aa); kilobases(kb); base pairs (bp); nucleotides (nt); intramuscular (i.m.);intraperitoneal (i.p.); subcutaneous (s.c.); and the like.

Example 1

The development and characterization of a new generation ofsemipermeable ultrafiltration membrane, the silicon nanopore membrane(SNM), designed with approximately 7 nm-wide slit-pores to providemiddle molecule selectivity by limiting passage of proinflammatorycytokines is shown. The use of convective transport with a pressuredifferential across the SNM overcomes the mass transfer limitationsassociated with diffusion through nanometer-scale pores. The SNMexhibited a hydraulic permeability of 130 ml/hr/m²/mmHg. Analysis ofsieving coefficients revealed 80% reduction in cytokines passage throughSNM under convective transport. SNM protected encapsulated islets frominfiltrating cytokines and retained islet viability over 6 hours andremained responsive to changes in glucose levels unlike non-encapsulatedcontrols. The concept involves using the pressure difference between theartery and vein to generate ultrafiltrate and drive transport ofglucose, insulin, and other small molecules through the SNM to supportfunction of encased islets while preventing passage of immunecomponents. SNM design and fabrication, followed by characterization ofits immunobarrier properties under cytokine challenge with convectivetransport, and assessment of SNM encapsulated islet viability andglucose-insulin response were developed. Specifically, hydraulicpermeability measurement and solute selectivity for SNM were determined.Mouse islets were encapsulated between SNM in a closed mock-loop fluidcircuit (FIG. 7) under simulated physiological pressure difference inthe presence of a cocktail of pro-inflammatory cytokines includingTNF-α, IL-1 β, and IFN-γ. Islet viability and glucose stimulated insulinproduction were evaluated to demonstrate the potential of SNM as anencapsulation material for islet immunoisolation under convectivetransport. Together, these data demonstrate the novel membraneexhibiting unprecedented hydraulic permeability and immune-protectionfor islet transplantation therapy.

1.1 Materials and Methods

Experimental Overview

SNM were fabricated to produce an active membrane area (6×6 mm)consisting of ˜10⁶ rectangular slit pores with ˜7 nm in width, 300 nm indepth, and 2 μm in length (FIG. 1). The surface of SNM was subsequentlymodified with polyethylene glycol (PEG) to minimize protein fouling. AllSNM membranes in this study were tested with an average pore size of ˜7nm. The transport of small solutes was first analyzed includingcytokines across a single SNM using a pressure-driven filtrationassembly (FIG. 8). To mimic the proposed bioartificial pancreas devicewith convective ultrafiltration under physiological pressure, aconstruction of a benchtop mock-loop circuit consisting of a three-layerflow cell with two enclosed SNM (FIG. 9), where the top, middle, andbottom compartments recapitulated the “artery”, “encapsulated isletchamber”, and “vein”, respectively. The percentage of cytokines,glucose, and insulin was subsequently characterized within the differentlocations of the mock-loop device. Finally, the viability andglucose-insulin response of the SNM-encapsulated mouse islets in themock-loop circuit with circulating cytokines were tested.

1.2 Substrate Preparation

1.2.1 Silicon Nanopore Membranes (SNM) Architecture and Fabrication

Silicon nanopore membranes (SNM) have been prototyped from siliconsubstrates by MEMS technology as previously reported with somemodifications (FIGS. 2A-I). Briefly, the process used the growth of athin SiO₂ (oxide) layer on 400 μm-thick double side polished (DSP)silicon wafers followed by a low pressure chemical vapor deposition(LPCVD) of polysilicon (˜500 nm). The wafers were then specificallypatterned, dry oxidized, wet etched, deposited with a second polysiliconlayer, and finally blanket etched until 400 nm of polysilicon remainedand the underlying vertical oxide layer was exposed. The verticalsacrificial oxide layer defined the critical nanoscale pore size of themembranes. The low temperature oxide (LTO) (˜1 μm) was deposited ontopolysilicon of the wafers to serve as the hard mask for membraneprotection. Deep reactive ion etching (DRIE) removed the backside ofeach window until membranes were disclosed. Eventually, the sacrificialoxide was etched away in 49% hydrofluoric acid (HF) during the finalstep of the fabrication process to leave behind open nanoscale slitpores. The wafers were subsequently cut into 1×1 cm chips with aneffective area of 6×6 mm² containing 1500 windows each, with a total of10⁶ pores per membrane. Each rectangular pore was 7 nm in width, 300 nmin depth, and 2 μm in length. All membranes were cleaned using aconventional “piranha” clean procedure, which involved a 20min-immersion in 3:1 sulfuric acid (H₂SO₄)/hydrogen peroxide (H₂O₂)mixture, followed by thorough rinses in deionized (DI) water. Images ofSNM were obtained using scanning electron microscope (SEM) (Leo 1550)(FIG. 1).

1.2.2 Surface Modification of SNM with poly(ethylene glycol) (PEG)

SNM were covalently modified with PEG using a previously reportedprotocol with some modifications to prevent protein fouling on themembrane surface. The technique used for PEG attachment involved asingle reaction step which covalently couples silicon surface silanolgroup (Si—OH) to a chain of PEG polymer through a trimethoxysilane groupforming a Si—O—Si—PEG sequence. Briefly, SNM were immersed in a solutionof 3 mM2[methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEGsilane)(Gelest: SIM6492.7) in toluene for 2 hr at 70° C. A series of extensivewashing steps involving toluene, ethanol, and DI water were used torinse away unbounded PEG residue.

1.2.3 Hydraulic Permeability for SNM Pore Size Characterization

An automated mass and pressure measurement system was utilized forcharacterizing liquid flow through the SNM under a tangential-flowfiltration operation. The pore size of the SNM can be related tofiltration flow parameters using

$\begin{matrix}{{h = \sqrt[3]{\frac{{12\;\mu\; l\; Q}\;}{{nw}\;\Delta\; P}}},} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$where h is pore width, H is the viscosity, 1 is the membrane thickness,Q is the volumetric flow rate, n is the number of pores per membrane, wis the pore length, and ΔP is the transmembrane pressure. To assemblethe overall system for SNM pore size characterization (FIG. 9), air wasapplied through a syringe pump (Sigma: Z675709) into a water reservoir.Water was circulated by a peristaltic pump (Masterflex: 07551-00)through a differential pressure transducer (Omega: PX429 015GI), a flowcell with enclosed membrane, and returned to the original waterreservoir. The flow cell was assembled with the SNM submerged underwater to remove air bubbles from all compartments. Specifically, amembrane was positioned with the polysilicon interface facing down witha customized silicone gasket positioned on top of the membrane, followedby the final placement of a filtrate chamber on top of the gasket. Allsections were fastened together and secured to the base withhand-tightened hex bolts until gasket was visibly compressed. Theultrafiltrate permeated through the membrane was routed to a liquidcollection container that rested on a precision mass balance (MettlerToledo: XS205). Measurements from the differential pressure transducerand the mass balance were automatically collected with a dataacquisition laptop. A typical membrane hydraulic permeability testconsisted of 5 ml/min flow rate and 4 pressure cycles (5, 1, 5, and 1psi) for durations of 150 s each.

Using the specifications for pore length, membrane thickness, and totalnumber of pores provided based on individual wafer designs, the averagepore size of SNM was calculated using Equation 1. All SNM membranes inthis study were surface-modified with PEG and exhibited an average poresize of ˜7 nm.

1.3 Assessment of SNM Immunoisolation in vitro

1.3.1 Membrane Sieving Coefficients Under Pressure-Driven Filtration

Fluid was circulated by a peristaltic pump through a circuit thatconsisted of a differential pressure transducer, a polycarbonate flowcell with enclosed SNM, a three-way valve, and a fluid reservoir (FIG.8). The flow cell consisted of two separate flow cell compartmentssandwiching a single SNM and silicone gasket. The top filtrate chamberrouted permeated ultrafiltrate to a liquid collection container, whereasthe base chamber was connected to a three-way valve. A solution of 3%bovine serum albumin (BSA) (Sigma: A-7030) was used to flush the entireloop prior to the experiment. Solution consisting of mouse cytokinesTNF-α (1000 U/ml) (Peprotech: 315-01A), IFN-γ (1000 U/ml) (Peprotech:315-05), IL-1β (50 U/ml) (Peprotech: 211-11B) 37, glucose (400 mg/dL)(Sigma-Aldrich: G8270), and insulin (150 mU/L) (Novo Nordisk:0169-1833-11) in a 3% BSA solution was then switched to the circuit at 5ml/min with a physiological pressure difference ˜2 psi 38. Ultrafiltratethat permeated through the SNM was collected at various time points forup to 6 hrs and analyzed with the enzyme-linked immunosorbent assays(ELISA) (BD Biosciences: 560478 & 558258; Thermo Pierce: EM2IL1B). Thesieving coefficients of solutes across SNM were calculated using

$\begin{matrix}{{S = \frac{C_{f}}{C_{b}}},} & \left( {{Equation}\mspace{14mu} 2} \right)\end{matrix}$where S is the sieving coefficient, Cf is the concentration of thesolute in the filtrate, and Cb is the molecule concentration in the bulkretentate solution.1.3.2 Solute Distribution in the Mock-Loop Circuit

A mock-loop circuit assembly with three flow cell components withoutcells to mimic the architecture of the final bioartificial pancreasdevice. Briefly, two SNM with customized silicone gasket frames weresandwiched in between three flow cell components. The middle flow cellwas the encapsulation chamber comprised of a cylindrical chamberseparating the two membranes. A peristaltic pump drove the fluid throughthe top of the flow cell mimicking the “artery”, then over the bottom ofthe flow cell resembling “vein”, and finally back to the originalreservoir. For convective experiments, a three-way valve was used tocreate flow resistance for a physiological pressure difference psibetween the top and the bottom compartments of the flow cell.Ultrafiltration occurred in the middle encapsulation chamber at thispressure difference. To study the transport of cytokines through thethree-layered bioartificial pancreas device, solution consisting ofmouse cytokines TNF-α (1000 U/ml), IFN-γ (1000 U/ml), and IL-1β (50U/ml), glucose (400 mg/dL), insulin (150 mU/L) in 3% BSA was circulatedthrough the circuit at a flow rate of 5 ml/min. Silicon membranes with1000 nm-wide slit pores (SμM) were used as the control. Solutions werecollected and analyzed with ELISA at the end of 6-hr experiments for thetop, middle, and bottom chambers.

1.3.3 Culture of Membrane-Encapsulated Islets in the Mock-Loop Circuit

All procedures described involving isolation of mouse islets wereperformed in accordance with protocols approved by the InstitutionalAnimal Care and Use Committee (IACUC) at the University of California,San Francisco (UCSF). Mouse islets were isolated from 8 to 10-week-oldmale B6 mice (Jackson Laboratories) based on previously describedprotocols 40. Harvested islets were maintained in suspension culturewith RPMI 1640 with L-glutamine and 11.1 mM glucose (Gibco: 11875-093),10% fetal bovine serum (FBS) (Gibco: 16000), and 1%penicillin-streptomycin (P/S) (UCSF Cell Culture Facility: CCFGK003). Agroup of 500 mouse islets were introduced into the middle encapsulationchamber of the mock-loop device (FIG. 7). To evaluate cell performancewith cytokine exposure, the circuit reservoir was replaced with culturemedium added with TNF-α (1000 U/ml), IFN-γ (1000 U/ml), and IL-1β (50U/ml) for 6 hrs. Static culture conditions with or without cytokineexposure were used as the controls. Mouse islets were subsequentlyisolated for viability testing and glucose challenge.

1.3.4 Islet Viability

Islet viability was assessed by double staining with fluoresceindiacetate (FDA) (Sigma: F7378) and propidium iodide (PI) (Sigma: 287075)as described by protocol (SOP Document: 3104, A02) from NationalInstitute of Allergy and Infectious Diseases (MAID). Briefly, mouseislets were incubated in phosphate buffered saline (PBS) containing0.067 HM FDA and 4.0 HM PI for 30 min and extensively washed in PBS toremove excess staining. Images of mouse islets were obtained using laserscanning Nikon Spectral Clsi confocal microscope (Nikon Instruments).Viability of islets was calculated based on the ratio between the numberof live cells in the islet and the area of that islet.

1.3.5 Glucose Stimulated Insulin Secretion Assay

Mouse islets retrieved from the middle chamber of the mock-loop circuitwere rested in RPMI 1640 containing 30 mg/dL glucose (Gibco: 11879) for15 minutes before exposed to medium containing 300 mg/dL glucose for 15minutes. After glucose stimulation, the islets were then returned tomedium containing 30 mg/dL glucose. Supernatant was collected every 5minutes during the series of incubations and insulin content wasmeasured with mouse insulin ELISA kits (Mercodia:10-1247-01) andnormalized by extracted total protein concentration (Thermo: 78505;23225).

1.4 Statistical Analysis

Sample pairs were analyzed using Student's t-test. Multiple samples wereevaluated with oneway or two-way analysis of variance (ANOVA) followedby Bonferroni and multiple comparison using Graphpad Prism software (SanDiego, Calif.). A p value of <0.05 was accepted as statisticallysignificant for all analyses.

1.5 Results

MEMS fabrication technologies offers unprecedented potential inreproducibility and precision to engineer controlled pore dimensionsthat can selectively block the passage of immune components whileallowing transport of small molecules (e.g. glucose and insulin) tosustain the viability of the encased cells. In the present study, thepermeability and selectivity of the SNM were characterized to preventcytokine infiltration and assessed the functional performance ofSNM-encapsulated mouse islets in a mock-loop device under convectivetransport.

1.5.1 SNM Design and Fabrication

A new generation of semipermeable membranes, SNM, with slit-pore designsinitially investigated by Desai et al. 26, 27. The SNM exhibit a poresize distribution of ˜1%, and a consistent pore size control in therange of 5-15 nm (FIG. 1) has been engineered. The slit poremicroarchitecture of SNM was achieved by dry oxidation of polysiliconfor the growth of silicon dioxide (SiO₂) (FIG. 2D) and through backsidepatterning with deep ion-reactive etching (DRIE) which resulted invertical sidewalls in each membrane window (FIG. 2H). This processallowed for fabrication of membranes with greater number of exposednanopores per area. The travel path could be further optimized bylowering the thickness of the membrane which can easily be controlled bythe thin film low-pressure chemical vapor deposition (LPCVD) (FIG. 2B)or dry etch process (FIG. 2G).

The utilization of a sacrificial layer to define the nanopores resultedin a membrane with a straight slit pore path that presents a shorterdistance for molecules to travel. The permeability-selectivity analysisfor ultrafiltration demonstrated that membranes with slit-shaped poresshowed higher performance and greater selectivity at a given value ofpermeability, than membranes with cylindrical pores for pore size below100 nm. To circumvent the slow concentration-dependent diffusionoccurred in size-restricted nanoporous membranes, the concept of usingconvection-dominated transport is advantageous in terms of creatingfaster solvent movement under transmembrane pressure gradient, whichefficiently drags small molecules such as glucose and insulin acrossmembranes to the encapsulated cells.

1.5.2 SNM Permeability and Selectivity Characterization

Permeability and selectivity of the SNM were characterized with thehydraulic permeability testing setup (FIG. 9), which uses liquid flowthrough planar nanoporous membranes under tangential-flow filtrationoperation. It was demonstrated that SNM with pore sizes of 7 nmgenerated a hydraulic permeability of 130 ml/hr/m²/mmHg, which is muchgreater compared with conventional polymer membranes (˜40 ml/hr/m²/mmHg)used in previous bioartificial pancreas devices. To further demonstratethe feasibility of SNM for immunoisolation, the membrane selectivity wascharacterized against transport of cytokines and small molecules usingthe pressure-driven ultrafiltration system (FIG. 8). Solute transportwas evaluated at ˜2 psi driving pressure to mimic the typicalphysiological pressure difference between artery and vein 38, whichresults in an ultrafiltration rate of ˜4 ul/min. The membrane Pecletnumber (Pe) for the pressure-driven ultrafiltration system wassignificantly greater than 1, suggesting that convective transportdominates. The observed sieving coefficients (calculated using Equation.2) should reflect the rejection characteristics of the membrane. After 6hours, the sieving coefficients of TNF-α, IFN-γ, and IL-1β were 0.16,0.27, and 0.27, respectively (FIG. 5). In contrast, the sievingcoefficients of glucose and insulin quickly reached 1 (FIG. 5). Thesedata collectively demonstrate that SNM provide about 80% rejection ofcytokine passage, while allowing complete transport of small molecules.Because concentration polarization and transmembrane diffusion werenegligible in this experimental system, the observed sieving coefficientshould be equal to the product of the solution partition coefficient (Φ)and the convective hindrance factor (Kc). Previously, Dechadilok andDeen derived an analytic expression for the product of ΦKc whichdescribes a rigid sphere passing in a slit shaped pore:ΦK_(C)=1−3.02λ²+5.776λ³−12.3675λ⁴+18.9775λ⁵−15.2185λ⁶+4.8525λ⁷  (Equation3),where λ is the relative solute size indicating the ratio between thediameter of the molecule and the width of slit-pore channel. Based onthe observed sieving coefficients of cytokines (FIG. 5), thecorresponding relative solute sizes λ from Deen's model (Eq. 3) can becalculated for TNF-α, IFN-γ, and IL-1β as 0.83, 0.74, and 0.74,respectively. The experimental relative solute sizes of these cytokinesare larger than the theoretic values, as indicated by Stokes-Einstein'sradius 14 (FIG. 10). This difference in relative solute sizes betweenthe experimental and theoretical values could be explained by the factthat cytokines are not strictly spherical: TNF-α is a packed cubic shapeconsisting of trimers formed with β-sheet structure, IFN-γ is a globulardimer with flattened elliptical shaped subunits 52, and IL-1β hasβ-strands wrapped around in a tetrahedron-like fashion. Furthermore, theelectrostatic interactions associated with diffuse electrical doublelayer (EDL) around charged proteins could also increase the overallmolecule size, thereby overestimating the experiment relative solutesizes.

In summary, the SNM enables higher levels of ultrafiltrate productionand demonstrate selective rejection against middle molecules likecytokines. Therefore, by encapsulating islets in SNM, it was postulatedthat the increased convective mass transport of nutrients and glucosecan support islet viability and insulin production, while the selectiverejection of immune components enables immunoisolation.

1.5.3 Assessment of SNM-Encapsulated Islets Cultured Under Mock-LoopCircuit

The feasibility of developing an implantable SNM-encapsulatedbioartificial pancreas device using convective transport wasdemonstrated using a mock-loop setup. The middle cell chamber issandwiched between two membranes to closely mimic the in vivo conditionswhere SNM-encapsulated islets will be mounted as an arterio-venous (AV)graft (FIG. 6). The pressure difference between the artery and vein willgenerate the ultrafiltrate and drive transport of water, salts, glucose,insulin, and other small molecules through the SNM, while passage ofimmune components such as cytokines will be blocked. After passing thecytokine-contained media from the reservoir through the mock-loopcircuit for 6 hr under applied physiological pressure ˜2 psi 38, samplesthat were collected from the top, middle, and bottom chambers of theflow cell device were compared against the reservoir concentration. Thelevel of cytokines TNF-α, IFN-γ, and IL-β were significantly reduced to30%, 35%, and 34% in the middle chamber, whereas small molecules insulinand glucose passed completely (˜100%) through both membranes (FIG. 11).To further examine the SNM-encapsulated islets under convectivetransport in the proposed mock-loop circuit, mouse islets were loadedinto the middle chamber with or without cytokine circulation for 6 hr.The static culture incubated with cytokines showed a more than 2.2-foldincrease in cell death compared to the static culture without cytokines,mock-loop device without cytokines, and mock-loop flow cell device withcytokines (FIG. 3). Moreover, no significant change in islet viabilitywas observed among the static culture without cytokines, mock-loopdevice without cytokines, and mock-loop flow cell device with cytokines(FIG. 3). This demonstrated the effectiveness of SNM to protect isletsfrom pro-inflammatory cytokine attack maintaining islet viability.

Additionally, the static culture without cytokines, mock-loop devicewithout cytokines, and mock loop flow cell device with cytokinesdemonstrated a 3.0-fold, 2.6-fold, and 4.1-fold changes, respectively,in the amount of insulin secreted during high glucose challenge comparedwith those secreted during low glucose challenge, respectively (FIG. 4).However, the static culture incubated with cytokines exhibited littlevariation in insulin secretion upon changes in glucose level (FIG. 4)due to loss in islet viability (FIG. 3). The glucose challengedemonstrated that the SNM-encapsulated mouse islets responded properlyto changes in glucose level, whereas cytokine-infiltrating mouse isletslost their insulin-secreting ability to sense glucose stimuli. Thesedata confirmed the usefulness of SNM to provide desired immunoisolationto support the viability and functional performance of the encapsulatedislets.

1.4 Conclusions

An improved silicon nanopore membrane, SNM, for the encapsulation ofpancreatic islets under convective flow was developed and characterized.The SNM structure was specifically designed to obtain a well-definedslit pore in the nanometer range with a remarkably high hydraulicpermeability. Furthermore, SNM achieved high molecule selectivityagainst middle molecules such as cytokines under convective transportand provided adequate immuneprotection to the encapsulated islets whilegenerating sufficient filtrate to support viability and functionality ofthe encapsulated islets.

Example 2

Semipermeable membrane capsules can immunoprotect transplanted islets byblocking passage of the host's immune components while providingexchange of glucose, insulin and other small molecules. However,capsules-based diffusive transport often exacerbates ischemic injury toislets by reducing the rate of oxygen and nutrient transport. Theefficacy of a newly-developed semipermeable ultrafiltration membrane,the silicon nanopore membrane (SNM) under convective-driven transport,limits the passage of pro-inflammatory cytokines while overcoming themass transfer limitations associated with diffusion throughnanometer-scale pores. SNM-encapsulated mouse islets perfused in culturesolution under convection outperformed those under diffusive conditionsin terms of magnitude (1.49-fold increase in stimulation index and3.86-fold decrease in shut-down index) and rate of insulin secretion(1.19-fold increase and 6.45-fold decrease during high and low glucosechallenges) respectively. SNM-encapsulated mouse islets under convectiondemonstrated rapid glucose-insulin sensing within a physiologicallyrelevant time-scale while retaining healthy islet viability even undercytokine exposure. The encapsulation of islets with SNM under convectionimproves graft function and survival.

This study showed that SNM and silicon micropore membrane (SμM) with 7nm and 1000 nm-wide slit-shaped pores respectively, were used toencapsulate mouse islets under diffusive and convective conditions withand without cytokine exposure. The islets were then exposed to varyingconcentration of glucose inside the reservoir culture medium, andglucose-stimulated insulin responses and islet viability were evaluated.In addition, to determine the immunoprotective effect of the membranes,a highly concentrated cocktail of pro-inflammatory cytokines was addedto the circulating system to challenge the encapsulated islets.

Materials and Methods

SNM were designed to have an active membrane area (6×6 mm) consisting of˜10⁶ rectangular slit pores with an average pore size of 7 nm in width,2 μm in length, and 300 nm in depth (FIG. 1). The surface of SNM wascoated with polyethylene glycol (PEG) to minimize protein fouling. AllSNM used in this study exhibited a measured average pore size of ˜7 nmpost-pegylation. The control silicon micropore membrane (SμM) had thesame design, but with an average pore size of 1000 nm. In this study, itwas observed how encapsulated islets responded to changes in glucoseconcentration across a single silicon membrane under convectivetransport (˜2 psi transmembrane pressure) or diffusive transport (0 psitransmembrane pressure) using a pressure-driven filtration circuit. Theglucose-insulin response was further tested by using highly concentratedcytokine solution in the circuit. The respective stimulation index (SI)and shut-down index (SDI) of encapsulated islets subsequently analyzedunder convective and diffusive conditions. The rate of change in insulinproduction was also tested based on the slopes of curves that werefitted on glucose-insulin kinetics graphs to describe the quickness ofinsulin being secreted as glucose concentration changes. Finally, theviability of encapsulated islets was characterized in thepressure-driven filtration assembly under various mass transfer andcytokine exposure conditions.

1.1 Substrate Preparation

1.1.1 Silicon Nanopore Membranes (SNM) and Silicon Micropore Membrane(SμM): architecture and Fabrication

Silicon nanopore membranes (SNM) have been prototyped from siliconsubstrates by MEMS technology as previously reported19, 36-37 with somemodifications (FIGS. 2A-I). Briefly, the process used the growth of athin SiO₂ (oxide) layer on 400 μm-thick double side polished (DSP)silicon wafers followed by a low pressure chemical vapor deposition(LPCVD) of polysilicon (˜500 nm). The wafers were then specificallypatterned, dry oxidized, wet etched, deposited with a second polysiliconlayer, and finally blanket-etched until 400 nm of polysilicon remainedand the underlying vertical oxide layer was exposed. The verticalsacrificial oxide layer defined the critical nanoscale pore size of themembranes. The low temperature oxide (LTO) (˜1 μm) was deposited ontopolysilicon of the wafers to serve as the hard mask for membraneprotection. Deep reactive ion etching (DRIE) removed the backside ofeach window until membranes were disclosed. Eventually, the sacrificialoxide was etched away in 49% hydrofluoric acid (HF) during the finalstep of the fabrication process to leave behind open nanoscale slitpores. The wafers were subsequently cut into 1×1 cm chips with aneffective area of 6×6 mm2 containing 1500 windows each, with a total of106 pores per membrane. Each rectangular pore was 7 nm in width, 300 nmin depth, and 2 μm in length. Silicon micropore membrane (SμM) werefabricated to produce wafer-scale arrays of 500 nm by 4 μm rectangularslit pores with 1000 nm-wide slit width using similar process. Thewafers were diced to form 1×1 cm chips with an effective area of 6×6 mm2containing 1500 windows each, with a total of 3.126 pores per membrane.All membranes were cleaned using a conventional “piranha” cleanprocedure, which involved a 20 min-immersion in 3:1 sulfuric acid(H2SO4)/hydrogen peroxide (H2O2) mixture, followed by thorough rinses indeionized (DI) water. Images of SNM were obtained using scanningelectron microscope (SEM) (Leo 1550) (FIGS. 2A-I).

1.1.2 Surface Modification of SNM with poly(ethylene glycol) (PEG)

SNM were covalently modified with PEG using a previously reportedprotocol38 with some modifications to prevent protein fouling on themembrane surface. The technique used for PEG attachment involved asingle reaction step which covalently couples silicon surface silanolgroup (Si—OH) to a chain of PEG polymer through a trimethoxysilane groupforming a Si—O—Si—PEG sequence. Briefly, SNM were immersed in a solutionof 3 mM 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEG-silane)(Gelest: SIM6492.7) in toluene for 2 hr at 70° C. A series of extensivewashing steps involving toluene, ethanol, and DI water were used torinse away unbounded PEG residue.

1.1.3 Hydraulic Permeability for SNM Pore Size Characterization

An automated mass and pressure measurement system was utilized forcharacterizing liquid flow through the SNM under a tangential-flowfiltration operation.9 The pore size of the SNM can be related tofiltration flow parameters using (Equation 1), where h is pore width, μis the viscosity, 1 is the membrane thickness, Q is the volumetric flowrate, n is the number of pores per membrane, w is the pore length, andΔP is the transmembrane pressure. To assemble the overall system for SNMpore size characterization (FIG. 9), air was applied through a syringepump (Sigma: Z675709) into a water reservoir. Water was circulated by aperistaltic pump (Masterflex: 07551-00) through a differential pressuretransducer (Omega: PX429 015GI), a flow cell with enclosed membrane, andreturned to the original water reservoir. The flow cell was assembledwith the SNM submerged under water to remove air bubbles from allcompartments. Specifically, a membrane was positioned with thepolysilicon interface facing down with a customized silicone gasketpositioned on top of the membrane, followed by the final placement of afiltrate chamber on top of the gasket. All sections were fastenedtogether and secured to the base with hand-tightened hex bolts until thegasket was visibly compressed. The ultrafiltrate permeated through themembrane was routed to a liquid collection container that rested on aprecision mass balance (Mettler Toledo: XS205). Measurements from thedifferential pressure transducer and the mass balance were automaticallycollected with a data acquisition laptop. A typical membrane hydraulicpermeability test consisted of 5 ml/min flow rate and 4 pressure cycles(5, 1, 5, and 1 psi) for durations of 150 s each. Using thespecifications for pore length, membrane thickness, and total number ofpores provided based on individual wafer designs, the average pore sizeof SNM was calculated using Equation 1. All SNM membranes in this studywere surface-modified with PEG and exhibited an average pore size of ˜7nm.

1.2 Culture of Membrane-Encapsulated Islets in the Pressure-DrivenFiltration Assembly

All procedures described involving isolation of mouse islets wereperformed in accordance with protocols approved by the InstitutionalAnimal Care and Use Committee (IACUC) at the University of California,San Francisco (UCSF). Mouse islets were isolated from 8 to 10-week-oldmale B6 mice (Jackson Laboratories) based on previously describedprotocols. Harvested islets were maintained in suspension culture withRPMI 1640 with L-glutamine and 11.1 mM glucose (Gibco: 11875-093), 10%fetal bovine serum (FBS) (Gibco: 16000), and 1% penicillin-streptomycin(P/S) (UCSF Cell Culture Facility: CCFGK003).

A mock-loop circuit was assembled with two flow cell components.Briefly, one SNM with customized silicone gasket frames were sandwichedin between two flow cell components. A group of 40-50 mouse islets wereintroduced into the bottom chamber separated by the SNM from thecirculating fluid (5 ml/min) in the top chamber. A peristaltic pumpdrove the fluid through the top of the flow cell component, and finallyback to the original medium reservoir. For convective experiments, athree-way valve was used to create flow resistance for a physiologicalpressure difference ˜2 psi between the top and the bottom compartmentsof the flow cell. The membrane Peclet number (Pe) for thepressure-driven ultrafiltration system was significantly greater than 1,suggesting that convective transport dominates. For diffusiveexperiments, no transmembrane pressure was induced and fluid stillcirculated throughout the system. To study the effects of cytokines onSNM-encapsulated islets, solution consisting of mouse cytokines TNF-α(2,000 U/ml), IFN-γ (1,000 U/ml), and IL-1β (10,000 U/ml) was added tothe original reservoir. Silicon membranes with 1 μm-wide slit pores(SμM) were used as the control with adjusted pressure (˜0.127 psi) andflow rate (˜20 μl/min) to produce similar amount of ultrafiltrate as theSNM in this mock-loop system. Naked mouse islets cultured under staticconditions were also used as controls.

1.2.1 Glucose Challenge in the Pressure-Driven Filtration Mock-LoopSystem

The membrane-encapsulated mouse islets in the mock-loop systems wereexposed to a series of low (1.6 mM), high (16.6 mM), and low (1.6 mM)glucose (Gibco: 11879) stimulation for 30 min each. Supernatant wassampled every 10 min from the bottom islet chamber during this series ofglucose challenge. For convective experiments, an ultrafiltrate rate of˜3.5 ul/min was observed for the SNM with ˜7 nm pore size and the sameultrafiltrate rate was obtained for the SμM with lowered transmembranemembrane pressure and system flow rate. For diffusive experiments, isletchambers were re-filled after individual sampling to ensure that thevolume of islet chamber was kept constant at all time. This stepminimized the any bubbles that might potentially be formed during theprocess which could hinder mass transfer within the system. Insulincontent was measured with mouse insulin enzyme-linked immunosorbentassay (ELISA) kits (Mercodia: 10-1247-01) with accounted dilutions andnormalized by extracted total protein concentration (Thermo: 78505;23225). Naked mouse islets were also challenged under static culturecondition as controls. About 7-10 μl chamber fluid per islet were usedin all cases.

1.2.2 Analysis of Stimulation Index (SI) and Shut-Down Index (SDI)

A stimulation index was calculated as the ratio of stimulated to basalinsulin secretion. In our study, the stimulation index (SI) was theratio of (1) the first insulin collection point in the high glucosephase to the last insulin collection point of the previous low glucosephase (Immediate Stimulation), and (2) the highest insulin secretion inthe high glucose phase to the last insulin collection point of theprevious low glucose phase (Maximum Stimulation). The shut-down index(SDI) was calculated as the ratio of (1) the first insulin collectionpoint in the subsequent low glucose phase to the last insulin collectionpoint in the high glucose phase (Immediate Shutdown), and (2) the lowestinsulin secretion in the subsequent low glucose phase to the lastinsulin collection point in the high glucose phase (Maximum Shutdown).The stimulation index indicates the magnitude of insulin released asstimulated by a higher concentration of glucose, whereas the shut-downindex reflects the magnitude of cessation in insulin production onceglucose concentration returns to normal.

1.2.3 Analysis of Rate of Change in Insulin Secretion

The rate of change in insulin secretion was calculated for thestimulation and shut-down phases. For the stimulation phase, a curve wasfitted on the glucose-insulin kinetic graph with the last point ofinsulin produced during low glucose exposure to the highest point ofinsulin produced during high glucose exposure. For the shut-down phase,a curve was fitted on the glucose-insulin kinetic graph with the lastpoint of insulin produced during high glucose exposure to the firstpoint of insulin produced during low glucose exposure. The rate ofchange was obtained by taking derivatives of those curves to study thequickness of insulin being secreted during changes in glucoseconcentration.

1.2.4. Islet Viability

Islet viability was assessed by double staining with live green and deadred solutions (Invitrogen: R37601). Briefly, mouse islets were incubatedin live green and dead red solutions for 15 min at room temperaturefollowed by extensive washes in PBS to remove excess staining. Images ofmouse islets were obtained using laser scanning Nikon Spectral C1siconfocal microscope (Nikon Instruments). Viability of islets wascalculated based on the percentage of live cells in the islets asdescribed by protocol on assessment of islet viability by fluorescentdyes from Department of Surgery Division of Transplantation atUniversity of Wisconsin-Madison.

1.3 Statistical Analysis

Sample pairs were analyzed using Student's t-test. Multiple samples wereevaluated with one-way or two-way analysis of variance (ANOVA) followedby Bonferroni and multiple comparison using Graphpad Prism software (SanDiego, Calif.). A p value of <0.05 was accepted as statisticallysignificant for all analyses.

Results and Discussion

A construction of a benchtop flow loop circuit consisting of a singlemembrane that separated islets from the circulating fluid (FIG. 7).Using this system, the kinetics of glucose-stimulated insulin secretionof SNM- and SμM-encapsulated islets was characterized under bothconvective and diffusive transport modalities. The effect of cytokineexposure was further analyzed to the function of SNM- andSμM-encapsulated islets by adding a highly concentrated cocktail ofpro-inflammatory cytokines including TNF-α, IL-1 β, and IFN-γ to thecircuit. The ability of membrane-encapsulated islets to secrete insulinupon changes in glucose concentration was characterized by: (1)computing the stimulation index (SI) and shut-down index (SDI) whichreflect the magnitude of stimulatory and shut-down insulin response as afunction of changes in glucose concentration, respectively; and (2)characterizing the rate of change in insulin secretion as the ambientfluid changed from low-to-high and high-to-low glucose concentrations.The viability of encapsulated-islets was also assessed in the mock-loopcircuit at the end of the various experimental conditions.

2.1 Membrane Fabrication and Characteristics

The slit pore microarchitecture of SNM is produced by dry oxidation ofpolysilicon for the growth of silicon dioxide (SiO₂) followed bybackside patterning with deep ion-reactive etching (DRIE) that producesvertical walls in each membrane window (FIG. 2A). The SNM wafer is dicedinto 1 cm×1 cm chips, each with an active membrane area (6×6 mm)consisting of ˜10⁶ rectangular slit pores with ˜7 nm width, 300 nmdepth, and 2 μm thickness (FIGS. 2B-C). Using similar fabricationtechniques, silicon micropore membranes (SμM) chips were produced, eachwith an active membrane area (6×6 mm) consisting of 3.12×10⁶ rectangularslit pores with ˜1000 nm in width, 500 nm in depth, and 4 μm in length(FIGS. 2D-E). Previously, it was demonstrated that that SNM with ˜7 nmpore size resulted in a 3.25-fold increase in hydraulic permeabilitycompared with conventional polymer membranes used in other bioartificialpancreas devices. Whereas the SμM allowed complete passage of allmolecules, SNM demonstrated size selectivity with an ˜80% rejection ofcytokine passage, while allowing complete transport of glucose andinsulin.

2.2 Kinetics of Glucose-Stimulated Insulin Secretion of EncapsulatedIslets

2.2.1 No Cytokine Exposure

A benchtop flow loop circuit incorporating membrane-encapsulated isletsunder applied physiological transmembrane pressure (FIG. 9) was used. Itwas observed how encapsulated islets responded to changes in glucoseconcentration across a single silicon membrane under convectivetransport (˜2 psi transmembrane pressure) or diffusive transport (0 psitransmembrane pressure) using this flow circuit. Unencapsulated isletscultured under static conditions were used as controls. Islets under allconditions reacted quickly to the high glucose concentration (16.6 mM)within the first 10 minutes by producing more insulin (40 minute timepoint; FIG. 13A). The unencapsulated islets under static culture andSNM-encapsulated islets under diffusion reached the peak of the response20 minutes after high glucose exposure, whereas insulin secretion of theSNM-encapsulated islets under convection continued to increase duringthe entire 30-minute duration of high glucose challenge (FIG. 13A). Thequick insulin response within 5-10 minutes of high glucose exposure wasconsistent with normal functioning islets releasing insulin in abiphasic manner (e.g. the first insulin phase appeared within 5-10minutes followed by a second sustained phase). Furthermore, thestimulation index (SI), calculated as the ratio of the first insulincollection in the high glucose phase to the last insulin collection inthe previous low glucose phase (Immediate Stimulation), were generallycomparable among naked islets under static conditions and theSNM-encapsulated islets under convection and diffusion cases, which were3.92±1.07, 6.38±0.44, and 5.62±1.51, respectively (FIG. 13B). However,when the highest level of insulin secretion from high glucose phase wasused to calculate the magnitude of stimulation (Maximum Stimulation),the naked islets under static conditions and SNM-encapsulated isletsunder convection and diffusion cases showed SI of 5.29±0.69, 8.92±1.35,5.97±1.16, respectively (FIG. 13B). The SI of SNM-encapsulated isletsunder convection showed a 1.49-fold increase than that under diffusion.

Once the circuit was switched back to low glucose concentration (1.6 mM)from 60 to 90 minutes, the SNM-encapsulated islets under convectionexhibited a rapid shutdown in insulin production whereas a gradualdecrease in insulin production occurred for the capsule under thediffusive mode. The shut-down index (SDI), calculated as the ratio ofthe first insulin collection in the subsequent low glucose phase to thelast insulin collection in the previous high glucose phase (ImmediateShutdown), showed that the amount of insulin that was secretedsignificantly decreased for SNM-encapsulated islets under convection(0.20±0.03) compared with the naked islets under static culture(0.59±0.17) and SNM-encapsulated islets under diffusion (0.93±0.19)(FIG. 13C). When the lowest level of insulin secretion from thesubsequent glucose phase was used to calculate the magnitude of shutdown (Maximum Shutdown), the SDI showed that the amount of secretedinsulin significantly decreased for SNM-encapsulated islets underconvection (0.11±0.02) compared with the naked islets under staticculture (0.40±0.09) and SNM-encapsulated islets under diffusion(0.42±0.11). The SDI of SNM-encapsulated islets under convection showeda 3.86-fold decrease compared to that under diffusion. The slow insulinactivation and delayed shut-down response associated with diffusivetransport is consistent with previous studies. The SNM-encapsulatedislets under convection showed the ability to quickly activate and ceaseinsulin production.

As illustrated in Table 1 of FIG. 18, the rate of change in insulinproduction was monitored when conditions transitioned from low-high tohigh-low glucose phases. The rates of change in insulin activation andcessation were on the same scale in the naked islets under staticculture as in the SNM-encapsulated islets under diffusion (0.86 and 0.84for the stimulation and −0.71 and −0.42 for deactivation, respectively;FIG. 18). The SNM-encapsulated islets under convection showed 1.16- and1.19-fold increase in the rate of glucose-stimulated insulin responseand 3.82- and 6.45-fold decrease in the rate of insulin shut-downcompared with the naked islets under static culture and SNM-encapsulatedislets under diffusion, respectively. In short, the magnitude ofglucose-stimulated insulin secretion was higher for SNM-encapsulatedislets under convection compared to the naked islets under staticculture and SNM-encapsulated islets under diffusion as indicated by theSI (FIG. 13B). The SNM-encapsulated islets under convection showed thefastest rate of insulin production (˜1 normalized insulin content min−1(×10−2)) and cessation (˜−2.7 normalized insulin content min−1 (×10−2))compared to the other two conditions (Table 1).

Further comparison with the silicon micropore membrane(SμM)-encapsulated islets under convection showed that pressure-drivenconvection yields faster mass transport as the pore size becomes larger(1 μm). The naked islets under static culture, SNM-encapsulated isletsunder convection, and SμM-encapsulated islets under convection allquickly released more insulin during high glucose exposure from 40 to 60minutes (FIG. 14A). Whereas the level of insulin plateaued in the nakedislets, the amount of secreted insulin increased in the SNM-encapsulatedislets under convection from 50 to 60 minutes. However, theSμM-encapsulated islets under convection showed a maximum level ofsecreted insulin at 50 minutes followed by an immediate concentrationdrop at 60 minutes. The difference in the glucose-insulin kineticsbetween SNM- and SμM-encapsulation under convection during high glucosechallenge can be explained by: (1) the variation in the ultrafiltrationrate produced by two different types of membranes despite efforts toadjust both membranes to obtain the same amount of ultrafiltrate(section 4.2); and (2) possible protein adsorption on the SNM19, 30 thatresulted in the lack of negative feedback inhibition of insulinsecretion31 due to additional fouling resistance. Furthermore, the SIindicating the magnitude of insulin secretion during pre-stimulation andstimulation (Immediate Stimulation) were higher for SNM- andSμM-encapsulation under convection compared to naked islets under staticconditions, which were 6.38±0.44, 6.44±1.41, and 3.92±1.07, respectively(FIG. 14B). When the highest amount of insulin secretion in the highglucose phase was used to calculate SI (Maximum Stimulation),SμM-encapsulation under convection (8.92±1.34) and SμM-encapsulationunder convection (11.8±1.64) showed significantly higher SI compared tonaked islets under static conditions (5.29±0.69). The SDI calculatedfrom the ratio of insulin secretion from post-stimulation andstimulation (Immediate Shutdown) for SNM- and SμM-encapsulation underconvection were 0.20±0.03 and 0.25±0.09, which showed a significantdecrease in the magnitude of insulin secreted during low glucoseexposure compared to the naked islets (0.59±0.17) (FIG. 14C). This trendwas also observed for SNM-encapsulation under convection (0.11±0.02),SμM-encapsulation under convection (0.11±0.01), and the naked islets(0.40±0.09) when the SDI was calculated based on the ratio of lowestinsulin secretion from post-stimulation and stimulation (MaximumShutdown) (FIG. 14C).

In addition, the SμM-encapsulated islets under convection showed thefastest rate of response when switching from low to high glucosecondition (3.15 normalized insulin content min−1 (×10⁻²)) to the high tolow glucose situation (−3.36 normalized insulin content min−1 (×10⁻²))(FIG. 18). The SμM-encapsulated islets under convection demonstrated3.66- and 3.15-fold increase in the rate of glucose-stimulated insulinresponse, and 4.73- and 1.24-fold decrease in rate of insulin shut-downcompared with the naked islets under static culture and SNM-encapsulatedislets under convection, respectively (FIG. 18). All rates of change ininsulin production and cessation were comparable among the naked isletsunder static culture, SNM-encapsulated islets under diffusion, andSμM-encapsulated islets under diffusion (FIG. 18). Noticeably,membrane-encapsulation under diffusive scenarios showed a slower insulinresponse when stimulated with high concentration of glucose (includingFIGS. 24A-C & FIGS. 25A-C). This could be due to the potential formationof boundary layer by adsorption of molecules in the nanoscale pores.19,30 Depending on the choice of membranes and methods to stimulate theislets (diffusion vs. convection), all experimental conditions had a SIranging from 2.89 to 6.44 (including FIGS. 24A-C & FIGS. 25A-C), whichis consistent of typical values (2 to 20) for healthy mouse islets.32Convective conditions with SNM- and SμM-encapsulation outperformed thepure diffusive scenarios during the glucose-insulin activation andshut-down phases. In particular, convective transport with SμMencapsulation demonstrated superior response in insulin activation whilethe insulin shut-down was observed to be similar for both SNM and SμMencapsulation under convection.

2.2.2 Cytokine Exposure

A highly concentrated solution of pro-inflammatory cytokines consistingof TNF-α, IFN-γ, and IL-1β was used to investigate how theglucose-insulin kinetics of SNM-encapsulated islets are influenced bycytokine exposure. When challenged with high glucose concentration,SNM-encapsulated islets under convection immediately secreted insulin tothe maximum level within first 10 minutes followed by a slight decreasein insulin secretion in the next 20 minutes (FIG. 15A). However,SNM-encapsulated islets under diffusion showed an incremental increasein insulin secretion during high glucose exposure. Although an increasein the insulin secretion level for the naked islets under static cultureduring the high glucose challenge was also observed, the maximum levelof insulin secreted was not as amplified as the other two conditions.Furthermore, the magnitude of insulin secretion during pre-stimulationand stimulation (Immediate Stimulation) was significantly differentamong the naked islets under static conditions, and SNM encapsulationunder convection and diffusion as indicated by the SI, which were2.98±0.06, 6.22±0.69, and 4.29±0.34, respectively (FIG. 15A). When thehighest amount of insulin secretion in the high glucose phase was usedto calculate SI (Maximum Stimulation), SNM-encapsulation underconvection (6.50±0.42) showed an increase in SI compared toSNM-encapsulation under convection (4.99±0.51) and naked islets understatic conditions (3.85±1.51) (FIG. 15B). As the circuit was switchedback to low glucose concentration, SNM-encapsulated islets underconvection showed the most significant drop in insulin secretioncompared to the naked islets and SNM encapsulation under diffusion (FIG.15A). The SDI calculated based on the ratio of insulin secretion frompost-stimulation and stimulation (Immediate Shutdown) for naked islets,and SNM encapsulation under convection and diffusion were 1.1±0.36,0.42±0.19, and 0.8±0.12, respectively (FIG. 15C). The similar trend wasobserved for SNM-encapsulation under convection (0.26±0.02),SNM-encapsulation under diffusion (0.57±0.15), and the naked islets(0.70±0.12) when the SDI was calculated based on the ratio of lowestinsulin secretion from post-stimulation and stimulation (MaximumShutdown) (FIG. 15C). Further analysis of the rate of change in insulinproduction from low to high glucose stimulation showed thatSNM-encapsulated islets under convection produced 2.89 normalizedinsulin content min−1 (×10−2), whereas naked islets under staticconditions and SNM-encapsulated islets under diffusion produced 0.22 and0.73 normalized insulin content min−1 (×10⁻²) (Table 2). The rate ofchange in insulin production from high to low glucose cessation ofSNM-encapsulated islets under convection was −1.76 normalized insulincontent min−1 (×10⁻²), whereas that of the naked islets under staticculture and SNM-encapsulated islets under diffusion were −0.092 and−0.32 normalized insulin content min−1 (×10⁻²). The SNM-encapsulatedislets under convection exhibited a 13.1- and 3.96-fold increase in therate of insulin production compared with naked islets and SNMencapsulation under diffusion respectively, when conditions were changedfrom low to high glucose exposure with cytokines. The SNM-encapsulatedislets under convection also demonstrated a 19.1- and 5.5-fold increasein the rate of shutting down insulin secretion from high to low glucoseconditions compared with naked islets and SNM encapsulation underdiffusion. In summary, the SNM-encapsulated islets under convectionexceeded both naked islets and SNM encapsulation under diffusion interms of the magnitude of insulin produced when stimulated with highlevel of glucose (FIGS. 15B-C) and the rate at which insulin wasproduced and ceased due to changes in glucose concentration (FIG. 19).

Unlike the SNM-encapsulated islets under convection in which the maximumlevel of insulin secreted within 10 minutes of high glucose challenge,SμM-encapsulation under convection showed a continuous rise in insulinsecretion and reached the highest peak within 30 minutes of high glucoseexposure (FIG. 16A). Moreover, SNM-encapsulated islets under convectionexhibited the largest magnitude of glucose-stimulated insulin secretionpossessing a SI value of 6.22±0.69, which was significantly higher thanthat for the SμM-encapsulation case with a SI value of 4.66±0.07(Immediate Stimulation) (FIG. 16B). When the highest amount of insulinsecretion in the high glucose phase was used to calculate SI (MaximumStimulation), SNM- and SμM-encapsulation under convection (6.50±0.42 &6.37±0.11) showed an increase in SI compared to naked islets understatic conditions (3.85±1.51) (FIG. 16B). However, the SDI for immediateshutdown of SNM- and SμM-encapsulated islets under convection weresimilar in which the SDI were 0.42±0.19 and 0.40±0.04, respectively(FIG. 16C). The same trend was observed when examining the SDI of SNM-and SμM-encapsulation under convection (0.26±0.02 & 0.28±0.04) and thenaked islets (0.70±0.12) where the SDI was calculated based on the ratioof lowest insulin secretion from post-stimulation and stimulation(Maximum Shutdown) (FIG. 16C). Further analysis of the rate of changesin insulin production was calculated for SμM-encapsulated islets underconvection which showed a 1.46-fold decrease and 1.61-fold increasecompared with SNM-encapsulated islets under convection in transitioningfrom low to high glucose stimulation and from high to low glucoseshut-down, respectively (FIG. 19). Noticeably, all diffusive conditionswith both SNM- and SμM-encapsulation showed reduction in the magnitudeof insulin produced as well as decline in the rate of insulin productioncompared to all convective scenarios (FIGS. 26A-C and FIGS. 27A-C, FIG.29). In summary, convective transport with SNM encapsulationdemonstrated better performance than SμM-encapsulation in terms of themagnitude of insulin produced and ceased during high and low glucosephases as indicated by SI and SDI factors under cytokine exposure (FIGS.15A-C and FIGS. 16A-C), while the rate of changes in insulin secretionwas similar between the two (FIG. 19).

Comparing previous conditions that were not subjected to cytokines, itwas observed that conditions with cytokine exposure had a slightdecrease in SI values (including FIGS. 26A-C and FIGS. 27A-C). Nosignificant difference in the magnitude of insulin secreted before andafter cytokine exposure for SNM-encapsulation under convection (SI(Immediate Stimulation): 6.38±0.44 and 6.23±0.69, respectively) (FIG.13B & FIG. 15B) was observed, while the SμM-encapsulation underconvection and naked islets under static culture all declined slightlyin their SI values (Immediate Stimulation): SμM-encapsulation underconvection dropped from 6.44±1.41 to 4.66±0.07 (FIG. 14B & FIG. 16B),and naked islets decreased from 3.92±1.06 to 2.98±0.06 (FIG. 13B & FIG.15B). The naked islets under static culture showed a higher SDI value(Immediate Shutdown) with cytokine exposure (0.59±0.17) (FIG. 13C) thanthe no-cytokine condition (1.1±0.36) (FIG. 15C), whereas SNM- andSμM-encapsulation under convection showed consistent SDI values(Immediate Shutdown) before and after cytokines were added (FIG. 13C,FIG. 14C, FIG. 15C; FIG. 16C). When switching from high to low glucoseconditions, the naked islets showed a large variation in the SDI value(Immediate Shutdown), indicating partial loss of islet regulatoryfunction with insulin. In contrast, both membrane-encapsulatedconditions showed sharp drop in insulin production once they wereswitched back to low glucose environment (FIGS. 15A-C, FIGS. 16A-C).Cytokines namely TNF-α, IFN-γ, and IL-1β are known to be synergisticallycytotoxic through a cascade of inflammatory events such as production ofnitric oxide and chemokines, and trigger of endoplasmic reticulum stressto cause loss of islet viability and functionality. It was speculatedthat cytokines damaged the naked islets as shown by their changes in SIand SDI values mentioned above, whereas the selectivity of the SNM andSμM membranes hindered cytokine infiltration and preserved isletfunction.

2.3 Islet Viability

In addition to the glucose-insulin kinetics of SNM- andSμM-encapsulation described above, the islet viability was investigatedto understand if cytokines caused excessive islet dysfunction (FIG. 6).The naked islets with cytokine exposure showed significantly more celldeath compared to all other groups including SNM- and SμM-encapsulationunder convection (FIG. 6,a). All membrane-associated diffusiveconditions showed normal health comparable to the untreated naked isletsunder static culture (FIG. S6). Some level of cytokine-induced deathdamage was observed in the SμM-encapsulation under convection as aresult of their inability to completely exclude cytokines likely due tothe large membrane pore size (FIG. 17A). However, the islet death in theSμM-encapsulation under convection was not as significant as in thecontrol scenario with naked islets. The SNM-encapsulated islets underconvection with cytokine exposure showed similar viability compared toSNM-encapsulating and healthy control conditions without cytokines.These observations confirm that membrane protection afforded by SNMprovides sufficient immunoisolation to support viability and functionalperformance of the encapsulated islets.

Conclusions

In this study, the glucose-insulin kinetics of an improved siliconnanopore membrane was characterized, SNM, for the encapsulation ofpancreatic islets under convective flow. The glucose-insulinresponsiveness of membrane-encapsulated islets was analyzed under aseries of low, high, and low glucose challenge by: (1) SI and SDIvalues, which show the magnitude of insulin secreted when transitioningfrom low to high glucose condition or vice versa; and (2) rate of changein insulin secretion, which indicates how quickly the system respondsfrom low to high glucose condition or vice versa. Based on theseparameters, it was found that convective mode performed better thandiffusive mode in both SNM and SμM encapsulations. In addition, onceexposed under cytokines, convective transport with SNM encapsulationdemonstrated superior performance over SμM encapsulation in terms of themagnitude of insulin produced and ceased during high and low glucosephases with healthy islet viability, while the rate of changes ininsulin secretion was on the same scale as that for the SμMencapsulation. In summary, SNM encapsulation under convective transportenables rapid glucose-insulin sensing to activate and cease insulinproduction based on the surrounding glucose concentration whileretaining healthy islet viability even under cytokine exposure. Our datademonstrates the importance of using convective transport to obtainfaster insulin activation and shut-down, which is a critical issue toaddress in many islet-encapsulating devices5, 35 with undesired delay inglucose-insulin response. Successful islet encapsulation with selectiveSNM under convective transport could potentially lower theimmunosuppressive drugs and their side effects resulted from currenttherapies, lead to the possibility of using xenogeneic or stem-cellderived cell sources to overcome donor shortage, and reduce dangerousepisodes of hypoglycemia for T1D patients in the future.

FIGS. 1A-B show silicon nanoporous membranes (SNM). FIG. 1A shows anoptical image of the SNM chip. FIG. 1B shows an SEM image of the surfaceof the membrane which illustrates nanopores with 2 um in length. FIG. 1Cshows an SEM image of the cross-section of the membrane whichillustrates one nanopore with 7 nm in width and 300 nm in depth.

FIGS. 2A-G show a schematic for fabrication of silicon nanoporemembranes. FIG. 2A shows piranha cleans of double side polished Siwafer. FIG. 2B shows thermal oxidation growth of SiO2 and low pressurechemical vapor deposition (LPCVD) of polysilicon. FIG. 2C shows dry-etchpatterning of polysilicon. FIG. 2D shows thermal oxidation growth ofSiO2 for use as sacrificial layer defining nanopores. FIG. 2E showspatterning of anchor layer by wet etch. FIG. 2F shows LPCVD ofpolysilicon. FIG. 2G shows blanket-etch of polysilicon until exposure ofvertical SiO₂ nanopores. FIG. 211 shows the deposition of lowtemperature oxide (LTO) for membrane protection and backside etch ofmembrane with deep reactive ion etching. FIG. 21 shows dry etch removalof LTO and wet etch release of SiO₂.

FIGS. 3A-D show in vitro viability of mouse islets under cytokineexposure. FIG. 3A shows viability of SNM-encapsulated mouse islets wasmeasured following the 6 hour experiment in which islets were subjectedto culture solution circulating the mock-loop circuit at 5 ml/min with apressure difference of 2 psi. FIG. 3B shows viable (green) and dead(red) cells were stained for control static culture (FIGS. 3A-B) andSNM-encapsulated mouse islets (FIGS. 3C-D). Experiments with cytokineexposure (indicated by +Ck) consisted of media containing TNF-α, IFN-γ,and IL-1β. The viability of islets was calculated based on the ratio ofdead cells (in red) over the islet area. Viabilities of islets in staticcultures were evaluated as control for comparison. SNM protectedencapsulated mouse islets from pro-inflammatory cytokines (SNM, +Ck),which showed similar viability to SNM-encapsulated mouse islets withoutcytokine exposure (SNM, −Ck) and control static culture without cytokineexposure (Control, −Ck). Control static culture with cytokine exposure(Control, +Ck) showed significantly more cell death compared with othergroups. (n>3, *p<0.05).

FIG. 4 shows glucose-stimulated insulin release of mouse islets in theSNM-encapsulation chamber and in static culture. Islets were subjectedto media containing low-glucose, high-glucose, and low-glucose for 15min each. Experiments with cytokine exposure (indicated by +Ck)consisted of culture solution containing TNF-α, IFN-γ, and IL-1β. Thestatic culture without cytokines (Control, −Ck), mock-loop devicewithout cytokines (SNM, −Ck), and mock-loop flow cell device exposedwith cytokines (SNM, +Ck) had a 3.0-fold, 2.6-fold, and 4.1-foldincrease in the amount of insulin secreted during high glucose challengeover those secreted during low glucose phase, respectively. However, thecontrol static culture with cytokine exposure (Control, +Ck) secretedlimited amount of insulin upon high glucose challenge due to the deadcells damaged by cytokine infiltration. (n>3, *p<0.05).

FIG. 5 shows a transport of various molecules through slit-pore of SNMunder a pressure difference of ˜2 psi. Sieving coefficients (S) wereexpressed as the ratio of the concentration of the filtrate over theconcentration of the feed (means±SE). BSA was used as a negativecontrol. Results showed that the sieving coefficients of TNF-α, IFN-γ,and IL-1β were 0.16, 0.27, and 0.27 after 6 hours, respectively. Thesieving coefficients of glucose and insulin quickly reached 1. Thesedata indicated that small molecules such as glucose and insulincompletely passed the SNM whereas the entry of cytokines was greatlyhindered under convective transport.

FIG. 6 shows a conceptual illustration of the implantable intravascularbioartificial pancreas device in the arm of a T1D patient. Transplantedislets will be encapsulated between two SNM sheets mounted on as anarterio-venous (AV) graft. The arterio-venous pressure differential willgenerate ultrafiltrate that continuously support the islets, which will,in turn, sense glucose levels and produce insulin that will be swpt intothe venous blood. The small pore size of the SNM ensures appropriateimmunoisolation between the transplanted islets and host.

FIG. 7 shows a schematic diagram of the mock-loop circuit for in vitroassessment of SNM-encapsulated islets under convective conditions. Aperistaltic pump circulated liquid through the top compartment of theflow cell, a pressure transducer, a 3-way valve, the bottom compartmentof the flow cell, and finally back to the original reservoir. The flowcell was composed of two membranes dividing the flow cell into threecompartments, where islets were placed inside the middle chamber.Ultrafiltrate flow occurred within the middle chamber between twosemipermeable membranes as the top membrane was adjacent to ahighpressure “arterial” blood channel and the second membrane wasadjacent to a low-pressure “vein” blood channel. The 3-way valve wasused to create a pressure difference of ˜2 psi between the top and thebottom compartment mimicking the physiological condition.

FIG. 8 shows a schematic diagram of the pressure-driven cytokinefiltration testing system. A peristaltic pump circulated liquid througha flow cell that connected to a 3-way valve to establish transmembranepressure. The permeated ultrafiltrate through the membrane was collectedat various time for up to 6 hrs.

FIG. 9 shows a schematic diagram of the hydraulic permeability testingsystem. Air was applied through a pressure regulator into the liquidreservoir. A peristaltic pump circulated this liquid through the flowcell with enclosed membrane. The flow cell connected to a differentialpressure transducer that was automatically controlled by a dataacquisition laptop to adjust the transmembrane pressure. The permeatedultrafiltrate was collected into a liquid container on top of aprecision mass balance. Data from the differential pressure transducerand the mass balance were automatically collected and stored in a dataacquisition laptop.

FIG. 10 shows a. comparison of relative solute size (λ). Experimentalrelative solute size (mean±SE) is calculated based on the sievingcoefficients for cytokines at 6 hrs. Theoretical values were determinedusing the Stokes-Einstein's equation 14.

FIG. 11 shows an assessment of solute distribution in the mock-loopsystem. The mock-loop circuit was composed of two membranes dividing theflow cell into the top, middle, and the bottom compartments.Concentration of solutes from each chamber was assessed at the end ofthe 6 hr experiment and was expressed as a percentage (mean±SE) relativeto that of the feed solution. Silicon micropore membrane (SHM) consistedof 1000 nm diameter slit pores were used as control. The data showedthat the amount of TNF-α, IFN-γ, and IL-1β were significantly reduced to30%, 35%, and 34% in the middle chamber, whereas small molecules insulinand glucose passed completely (˜100%) through SNM under convective flow.However, all molecules including cytokines passed into the middlechamber that were sandwiched between SHM. (n>3, *p<0.05).

FIG. 12A shows an SEM image of the tilted membrane surface which depictsnanopores with 2 μm in length. FIG. 12B shows an SEM image of thecross-section of the membrane which depicts nanopores with 7 nm in widthand 300 nm in depth. FIG. 12C shows an SEM image of the membrane surfacewhich depicts micropores with 4 μm in length. FIG. 12D shows an SEMimage of the cross-section of the membrane which depicts micropores with1 μm in width.

FIGS. 13A-C shows Glucose-insulin kinetics of SNM-encapsulated isletsunder convection and diffusion without cytokine exposure. FIG. 13A showsinsulin release kinetics of SNM-encapsulated mouse islets during90-minute low-high-low (1.6 mM, 16.6 mM, 1.6 mM) glucose stimulationunder convective (2 psi) (Cony) and diffusive transport (Diff) withoutsubjection to cytokines. The naked islets cultured under staticconditions were served as controls (Control). The SNM-encapsulatedislets under convective transport (SNM, Cony) exhibited higher insulinsecretion following stimulation at high glucose concentration and fasterinsulin release kinetics in response compared to those under diffusivetransport (SNM, Diff). (Mean±SEM, n>3). FIG. 13B shows the stimulationindex (SI) was calculated as the ratio of (1) the first insulincollection in the high glucose phase at 40 minutes to the last insulincollection point of the previous low glucose phase at 30 minutes(Immediate Stimulation), and (2) the highest insulin secretion in thehigh glucose phase to the last insulin collection point of the previouslow glucose phase at 30 minutes (Maximum Stimulation). The SI indicatesthe magnitude of insulin released as stimulated by a higherconcentration of glucose. Without cytokine exposure, SNM-encapsulatedislets under convection (SNM, Cony) and diffusion (SNM, Diff) inaddition to the naked islets cultured under static conditions (Control)all exhibited similar magnitude of glucose-induced insulin secretionwhen transitioning from low glucose to high glucose (ImmediateStimulation). However, the SI of SNM-encapsulated islets underconvection (SNM, Cony) was the highest compared to that under diffusion(SNM, Diff) and the naked islets cultured under static conditions(Control) when the highest insulin secretion in the high glucose phasewas used (Maximum Stimulation). (Mean±SEM, n≥3). FIG. 13C shows that theshut-down index (SDI) was the ratio of (1) the first insulin collectionpoint in the subsequent low glucose phase at 70 minutes to the lastinsulin collection point in the high glucose phase at 60 minutes(Immediate Shutdown), and (2) the lowest insulin secretion in thesubsequent low glucose phase to the last insulin collection point in thehigh glucose phase at 60 minutes (Maximum Shutdown). The SDI reflectsthe magnitude of cessation in insulin production once glucoseconcentration returns to normal. Without cytokine exposure,SNM-encapsulated islets under convection (SNM, Cony) exhibited thehighest magnitude of insulin reduction compared to the diffusivecondition (SNM, Diff) and the naked islet culture (Control) as glucosedropped low (Immediate Shutdown & Maximum Shutdown). (Mean±SEM, n≥3,*p<0.05).

FIGS. 14A-C shows glucose-insulin kinetics of SNM- and Sμm-enapsulatedislets under convection without cytokine exposure. FIG. 14A showsinsulin release kinetics of SNM- and SμM-encapsulated mouse isletsduring 90-minute low-high-low (1.6 mM, 16.6 mM, 1.6 mM) glucosestimulation under convective (2 psi) (Cony) without subjection tocytokines. The naked islets cultured under static conditions were servedas controls (Control). Without cytokine exposure, the SμM-encapsulatedislets under convective transport (SμM, Cony) exhibited higher insulinsecretion following stimulation at high glucose concentration and fasterinsulin release kinetics in response to glucose compared to theSNM-encapsulated islets under convective transport (SNM, Conv).(Mean±SEM, n≥3). FIG. 14B shows that the stimulation index (SI) wascalculated as the ratio of (1) the first insulin collection in the highglucose phase at 40 minutes to the last insulin collection point of theprevious low glucose phase at 30 minutes (Immediate Stimulation), and(2) the highest insulin secretion in the high glucose phase to the lastinsulin collection point of the previous low glucose phase at 30 minutes(Maximum Stimulation). The SI indicates the magnitude of insulinreleased as stimulated by a higher concentration of glucose. Withoutcytokine exposure, the SNM- and SμM-encapsulated islets under convection(SNM, Cony & SμM, Cony) all showed a higher magnitude of secretedinsulin compared to the naked islets cultured under static conditions(Control). Furthermore, the SI of SμM-encapsulated islets underconvection (SμM, Cony) was the greatest compared to that for the SNM(SNM, Cony) and naked islets cultured under static conditions (Control)when the highest insulin secretion in the high glucose phase was used(Maximum Stimulation). (Mean±SEM, n≥3). FIG. 14C shows that theshut-down index (SDI) was the ratio of (1) the first insulin collectionpoint in the subsequent low glucose phase at 70 minutes to the lastinsulin collection point in the high glucose phase at 60 minutes(Immediate Shutdown), and (2) the lowest insulin secretion in thesubsequent low glucose phase to the last insulin collection point in thehigh glucose phase at 60 minutes (Maximum Shutdown). The SDI reflectsthe magnitude of cessation in insulin production once glucoseconcentration returns to normal. Without cytokine exposure, both SNM-and SμM-encapsulated islets under convection (SNM, Cony & SμM, Cony)exhibited significant magnitude of insulin reduction compared to theislets cultured under static conditions (Control) once glucose droppedback low (Immediate Shutdown & Maximum Shutdown). (Mean±SEM, n≥3,*p<0.05).

FIGS. 15A-C shows glucose-insulin kinetics of SNM-encapsulated isletsunder convection and diffusion with cytokine exposure. FIG. 15A showsinsulin release kinetics of SNM-encapsulated mouse islets during90-minute low-high-low (1.6 mM, 16.6 mM, 1.6 mM) glucose stimulationunder convective (2 psi) (Cony) and diffusive transport (Diff) withsubjection to cytokines (+Ck). Experiments with cytokine exposure (+Ck)consisted of media containing TNF-α (2,000 U/mL), IFN-γ (1,000 U/mL),and IL-1β (10,000 U/mL). The naked islets cultured under staticconditions served as controls (Control, +Ck). The SNM-encapsulatedislets under convective transport (SNM, Cony, +Ck) exhibited higherinsulin secretion following stimulation at high glucose concentrationand faster insulin release kinetics in response compared to those underdiffusive transport (SNM, Diff, +Ck) and naked islets cultured understatic conditions (Control, +Ck). (Mean±SEM, n≥3). FIG. 15B shows thestimulation index (SI) was calculated as the ratio of (1) the firstinsulin collection in the high glucose phase at 40 minutes to the lastinsulin collection point of the previous low glucose phase at 30 minutes(Immediate Stimulation), and (2) the highest insulin secretion in thehigh glucose phase to the last insulin collection point of the previouslow glucose phase at 30 minutes (Maximum Stimulation). The SI indicatesthe magnitude of insulin released as stimulated by a higherconcentration of glucose. With cytokine exposure (+Ck), all conditionsincluding SNM-encapsulated islets under convection (SNM, Cony) anddiffusion (SNM, Diff), and the naked islets cultured under staticconditions (Control) exhibited varying level of magnitude inglucose-induced insulin secretion (Immediate Stimulation). However, whenusing the highest insulin secretion in the high glucose phase (MaximumStimulation), the calculated SI was the highest for SNM-encapsulatedislets under convection (SNM, Cony) compared to that under diffusion(SNM, Diff) and naked islets cultured under static conditions (Control).(Mean±SEM, n≥3, *p<0.05). FIG. 15C shows the shut-down index (SDI) thatwas calculated as the ratio of (1) the first insulin collection point inthe subsequent low glucose phase at 70 minutes to the last insulincollection point in the high glucose phase at 60 minutes (ImmediateShutdown), and (2) the lowest insulin secretion in the subsequent lowglucose phase to the last insulin collection point in the high glucosephase at 60 minutes (Maximum Shutdown). The SDI reflects the magnitudeof cessation in insulin production once glucose concentration returns tonormal. With cytokine exposure (+Ck), the SNM-encapsulated islets underconvection (SNM, Cony) exhibited the highest magnitude of insulinreduction compared to the diffusive condition (SNM, Diff) and the nakedislet culture (Control) as glucose dropped low (Immediate Shutdown &Maximum Shutdown). (Mean±SEM, n≥3, *p<0.05).

FIGS. 16A-C show glucose-insulin kinetics of SNM- and SμM-encapsulatedislets under convection with cytokine exposure. FIG. 16A shows Insulinrelease kinetics of SNM- and SμM-encapsulated mouse islets during90-minute low-high-low (1.6 mM, 16.6 mM, 1.6 mM) glucose stimulationunder convective (2 psi) (Cony) with subjection to cytokines (+Ck). Thenaked islets cultured under static conditions served as controls(Control, +Ck). Experiments with cytokine exposure (+Ck) consisted ofmedia containing TNF-α (2,000 U/mL), IFN-γ (1,000 U/mL), and IL-1β(10,000 U/mL). With cytokine exposure (+Ck), the SμM-encapsulated isletsunder convective transport (SμM, Cony) exhibited a continuous insulinsecretion following stimulation at high glucose concentration from 40minutes to 60 minutes, while the SNM-encapsulated islets underconvection (SNM, Cony) showed a plateau in insulin production duringthis period of challenge. (Mean±SEM, n≥3). FIG. 16B shows thestimulation index (SI) was calculated as the ratio of (1) the firstinsulin collection in the high glucose phase at 40 minutes to the lastinsulin collection point of the previous low glucose phase at 30 minutes(Immediate Stimulation), and (2) the highest insulin secretion in thehigh glucose phase to the last insulin collection point of the previouslow glucose phase at 30 minutes (Maximum Stimulation). The SI indicatesthe magnitude of insulin released as stimulated by a higherconcentration of glucose. With cytokine exposure (+Ck), the SNM- andSμM-encapsulated islets under convection (SNM, Cony & SμM, Cony) and thenaked islet culture under static conditions (Control) all showed asignificant difference in the magnitude of insulin secreted upon highglucose challenge (Immediate Stimulation). However, the SNM- andSμM-encapsulated islets under convection (SNM, Cony & SμM, Cony) showedgreater difference in the magnitude of insulin secreted upon highglucose challenge when the highest insulin secretion was used (MaximumStimulation). (Mean±SEM, n≥3, *p<0.05). FIG. 16C shows that theshut-down index (SDI) was the ratio of (1) the first insulin collectionpoint in the subsequent low glucose phase at 70 minutes to the lastinsulin collection point in the high glucose phase at 60 minutes(Immediate Shutdown), and (2) the lowest insulin secretion in thesubsequent low glucose phase to the last insulin collection point in thehigh glucose phase at 60 minutes (Maximum Shutdown). The SDI reflectsthe magnitude of cessation in insulin production once glucoseconcentration returns to normal. With cytokine exposure (+Ck), the SNM-and SμM-encapsulated islets under convection (SNM, Cony & SμM, Cony)exhibited the highest magnitude of insulin reduction compared to thenaked islet culture (Control) as glucose dropped low (Immediate Shutdown& Maximum Shutdown). (Mean±SEM, n≥3, *p<0.05).

FIGS. 17A-B show in-vitro viability of mouse islets. FIG. 17A showsviability of mouse islets was measured following the 90-minutelow-high-low (1.6 mM, 16.6 mM, 1.6 mM) glucose stimulation in whichislets were subjected to the mock-loop circuit with (+Ck) or withoutcytokine exposure for SNM- and SμM-encapsulation under convection (SNM,C & SμM, C). The naked islet culture under static culture with cytokineexposure (Control, +Ck) showed significantly less viability compared toall other conditions. (Mean±SEM, n≥3, *p<0.05). FIG. 17B shows viable(green) and dead (red) cells were stained for control static culturewithout cytokines (A: Control), control static culture with cytokines(B: Control, +Ck), SNM-encapsulated mouse islets under convectionwithout cytokines (C: SNM, C), SNM-encapsulated mouse islets underconvection with cytokines (D: SNM, C, +Ck), SμM-encapsulated mouseislets under convection without cytokines (E: SμM, C), andSμM-encapsulated mouse islets under convection with cytokines (F: SμM,C, +Ck). Experiments with cytokine exposure (indicated by +Ck) consistedof media containing TNF-α, IFN-γ, and IL-1β. Both control static culturewith cytokines (B: Control, +Ck) and SμM-encapsulated mouse islets underconvection with cytokines (F: SμM, C, +Ck) showed a higher level ofislet damage compared to other groups, however, the viability ofSμM-encapsulated mouse islets under convection with cytokines (F: SμM,C, +Ck) was not statistically significant (n.s.) (FIG. 17A).

FIG. 18 shows the rate of change in insulin secretion without cytokineexposure in Table 1. The rate of change in insulin production wascalculated based on the slopes of curves that were fitted onglucose-insulin kinetics graphs to describe the quickness of insulinbeing secreted as glucose concentration changes. Without subjection tocytokines, SμM-encapsulated mouse islets under convection (SμM, Cony)showed the fastest response following high glucose exposure while SNM-and SμM-encapsulated mouse islets under convection (SNM, Cony & SμM,Cony) exhibited similar rate of insulin cessation when glucoseconcentration returned to normal.

FIG. 19 shows the rate of change in insulin secretion with cytokineexposure in Table 2. The rate of change in insulin production wascalculated based on the slopes of curves that were fitted onglucose-insulin kinetics graphs to describe the quickness of insulinbeing secreted as glucose concentration changes. With subjection tocytokines (+Ck), SNM-encapsulated mouse islets under convection (SNM,Cony) showed the fastest response following high glucose exposure whileSNM- and SμM-encapsulated mouse islets under convection (SNM, Cony &SμM, Cony) exhibited similar rate of insulin cessation when glucoseconcentration returned to normal.

FIGS. 20A-D shows an illustration of the process and fixtures for CellScaffold construction. FIG. 20A presents the laser cut acrylic sheet ontop of the silicone wire holder. FIG. 20B shows red wires in place tocreate the ultrafiltrate channels. FIG. 20C presents the purple isletagarose gel poured into the laser cut void of the acrylic sheet. FIG.20D illustrates the completed Cell Scaffold after removal of red wiresand silicone wire holders.

FIG. 21 shows a zoomed-in view of the components of the bioartificialdevice.

FIG. 22 shows the inlet and outlet components of the bioartificialdevice. The assembled iBAP is used for both in vitro and in vivoexperiments.

FIGS. 23A-B shows an illustration of the bioartificial device connectedinline to an arterial-venous graft and an ultrafiltrate catheterdelivering insulin rich ultrafiltrate to a vein. FIG. 23B shows across-sectional view along the axial and perpendicular directions offlood flow illustrating a single blood channel surrounded by the SNM(green) encapsulated IC (blue) on both sides. Ultrafiltrate crosses theSNM encapsulated inlet chamber into the ultrafiltrate chamber and thenflows along the ultrafiltrate flow path to the ultrafiltrate outlet.

FIGS. 24A-C show glucose-insulin kinetics of SμM-encapsulated isletsunder convection and dissufsion without cytokine exposure. FIG. 24Ashows insulin release kinetics of SμM-encapsulated mouse islets during90-minute low-high-low (1.6 mM, 16.6 mM, 1.6 mM) glucose stimulationunder convective (2 psi) (Cony) and diffusive transport (Diff) withoutsubjection to cytokines. The naked islets cultured under staticconditions served as controls (Control). The SμM-encapsulated isletsunder convective transport (SμM, Cony) exhibited higher insulinsecretion following stimulation at high glucose concentration and fasterinsulin release kinetics in response compared to those under diffusivetransport (SμM, Diff). (Mean±SEM, n≥3). FIG. 24B shows the stimulationindex (SI) was calculated as the ratio of (1) the first insulincollection in the high glucose phase at 40 minutes to the last insulincollection point of the previous low glucose phase at 30 minutes(Immediate Stimulation), and (2) the highest insulin secretion in thehigh glucose phase to the last insulin collection point of the previouslow glucose phase at 30 minutes (Maximum Stimulation). The SI indicatesthe magnitude of insulin released as stimulated by a higherconcentration of glucose. Without cytokine exposure, SμM-encapsulatedislets under convection (SμM, Cony) and diffusion (SμM, Diff) inaddition to the naked islets cultured under static conditions (Control)all exhibited similar magnitude of glucose-induced insulin secretion(Immediate Stimulation). However, the SμM-encapsulated islets underconvection (SμM, Cony) showed the highest magnitude of insulin secretedwhen the highest insulin secretion in the high glucose phase was used(Maximum Stimulation). (Mean±SEM, n≥3). FIG. 24C shows the shut-downindex (SDI) was the ratio of (1) the first insulin collection point inthe subsequent low glucose phase at 70 minutes to the last insulincollection point in the high glucose phase at 60 minutes (ImmediateShutdown), and (2) the lowest insulin secretion in the subsequent lowglucose phase to the last insulin collection point in the high glucosephase at 60 minutes (Maximum Shutdown). The SDI reflects the magnitudeof cessation in insulin production once glucose concentration returns tonormal. Without cytokine exposure, SμM-encapsulated islets underconvection (SμM, Cony) exhibited the highest magnitude of insulinreduction compared to the diffusive condition (SμM, Diff) and the nakedislet culture (Control) as glucose dropped low (Immediate Shutdown).When the lowest insulin secretion in the low glucose phase was used,SμM-encapsulated islets under convection (SμM, Cony) also showed thelargest magnitude of insulin reduction (Maximum Shutdown). (Mean±SEM,n≥3, *p<0.05).

FIGS. 25A-C show glucose-insulin kinetics of SNM- and SμM-encapsulatedunder diffusion without cytokine exposure. FIG. 25A shows insulinrelease kinetics of SNM- and SμM-encapsulated mouse islets during90-minute low-high-low (1.6 mM, 16.6 mM, 1.6 mM) glucose stimulationunder diffusion (2 psi) (Diff) without subjection to cytokines. Thenaked islets cultured under static conditions served as controls(Control). Without cytokine exposure, SμM-encapsulated islets underdiffusive transport (SμM, Diff) exhibited higher insulin secretion thatslowly plateaued following stimulation at high glucose concentrationcompared to the SNM-encapsulated islets under diffusive transport (SNM,Diff). (Mean±SEM, n≥3). FIG. 25B shows the stimulation index (SI) wascalculated as the ratio of (1) the first insulin collection in the highglucose phase at 40 minutes to the last insulin collection point of theprevious low glucose phase at 30 minutes (Immediate Stimulation), and(2) the highest insulin secretion in the high glucose phase to the lastinsulin collection point of the previous low glucose phase at 30 minutes(Maximum Stimulation). The SI indicates the magnitude of insulinreleased as stimulated by a higher concentration of glucose. Withoutcytokine exposure, the SNM- and SμM-encapsulated islets under diffusion(SNM, Diff & SμM, Diff) all showed a similar magnitude of insulinsecretion compared with the naked islets cultured under staticconditions (Control) (Immediate Stimulation). Moreover, theSNM-encapsulated islets under diffusion (SNM, Diff) and naked isletscultured under static conditions showed an increase in SI compared tothe SμM-encapsulated islets under diffusion (SμM, Diff) when the highestinsulin secretion in the high glucose phase was used (MaximumStimulation). (Mean±SEM, n≥3). FIG. 25C shows the shut-down index (SDI)was the ratio of (1) the first insulin collection point in thesubsequent low glucose phase at 70 minutes to the last insulincollection point in the high glucose phase at 60 minutes (ImmediateShutdown), and (2) the lowest insulin secretion in the subsequent lowglucose phase to the last insulin collection point in the high glucosephase at 60 minutes (Maximum Shutdown). The SDI reflects the magnitudeof cessation in insulin production once glucose concentration returns tonormal. Without cytokine exposure, SNM- and SμM-encapsulated isletsunder diffusion (SNM, Diff & SμM, Diff) exhibited similar magnitude ofinsulin reduction compared to the islets cultured under staticconditions (Control) once glucose dropped back low (Immediate Shutdown).However, the level of shut down was more significant in SμM-encapsulatedislets under diffusion (SμM, Diff) than in the other two conditions(SNM, Diff & Control) when the lowest insulin secretion was used(Maximum Shutdown). (Mean±SEM, n≥3, *p<0.05).

FIGS. 26A-C show glucose-insulin kinetics of SμM-encapsulated isletsunder convection and diffusion with cytokine exposure. FIG. 26A showsinsulin release kinetics of SμM-encapsulated mouse islets during90-minute low-high-low (1.6 mM, 16.6 mM, 1.6 mM) glucose stimulationunder convective (2 psi) (Cony) and diffusive transport (Diff) withsubjection to cytokines (+Ck). Experiments with cytokine exposure (+Ck)consisted of media containing TNF-α (2,000 U/mL), IFN-γ (1,000 U/mL),and IL-1β (10,000 U/mL). The naked islets cultured under staticconditions served as controls (Control, +Ck). The SμM-encapsulatedislets under convective transport (SμM, Cony, +Ck) exhibited higherinsulin secretion and faster insulin release kinetics in response tostimulation at high glucose concentration compared to those underdiffusive transport (SμM, Diff, +Ck) and naked islets cultured understatic conditions (Control, +Ck). (Mean±SEM, n≥3). FIG. 26B shows thestimulation index (SI) was calculated as the ratio of (1) the firstinsulin collection in the high glucose phase at 40 minutes to the lastinsulin collection point of the previous low glucose phase at 30 minutes(Immediate Stimulation), and (2) the highest insulin secretion in thehigh glucose phase to the last insulin collection point of the previouslow glucose phase at 30 minutes (Maximum Stimulation). The SI indicatesthe magnitude of insulin released as stimulated by a higherconcentration of glucose. With cytokine exposure (+Ck), all conditionsincluding SμM-encapsulated islets under convection (SμM, Cony) anddiffusion (SμM, Diff), and the naked islets cultured under staticconditions (Control) exhibited varying level of magnitude inglucose-induced insulin secretion (Immediate Stimulation). TheSμM-encapsulated islets under convection (SμM, Cony) and naked isletscultured under static conditions (Control) showed an increase in themagnitude of insulin secretion when the highest insulin secretion in thehigh glucose phase was used (Maximum Stimulation). (Mean±SEM, n≥3,*p<0.05). FIG. 26C shows the shut-down index (SDI) was the ratio of (1)the first insulin collection point in the subsequent low glucose phaseat 70 minutes to the last insulin collection point in the high glucosephase at 60 minutes (Immediate Shutdown), and (2) the lowest insulinsecretion in the subsequent low glucose phase to the last insulincollection point in the high glucose phase at 60 minutes (MaximumShutdown). The SDI reflects the magnitude of cessation in insulinproduction once glucose concentration returns to normal. With cytokineexposure (+Ck), the SμM-encapsulated islets under convection (SμM, Cony)and under diffusion (SμM, Diff) both exhibited the highest magnitude ofinsulin reduction compared to the naked islet culture (Control) asglucose dropped low (Immediate Shutdown & Maximum Shutdown). (Mean±SEM,n≥3, *p<0.05).

FIGS. 27A-C show glucose-insulin kinetics of SNM- and SμM-encapsulatedislets under diffusion with cytokine exposure. FIG. 27A shows insulinrelease kinetics of SNM- and SμM-encapsulated mouse islets during90-minute low-high-low (1.6 mM, 16.6 mM, 1.6 mM) glucose stimulationunder diffusion (Diff) with subjection to cytokines (+Ck). The nakedislets cultured under static conditions served as controls (Control,+Ck). Experiments with cytokine exposure (+Ck) consisted of mediacontaining TNF-α (2,000 U/mL), IFN-γ (1,000 U/mL), and IL-1β (10,000U/mL). With cytokine exposure (+Ck), the SμM-encapsulated islets underdiffusive transport (SμM, Diff) exhibited the fastest insulin secretionat high glucose concentration from 40 minutes to 60 minutes followed bythe SNM-encapsulated islets under diffusion (SNM, Diff). The level ofglucose-induced insulin secretion from the naked islets cultured understatic conditions (Control) was not as significant as the other twogroups. (Mean±SEM, n≥3). FIG. 27B shows the stimulation index (SI) wascalculated as the ratio of (1) the first insulin collection in the highglucose phase at 40 minutes to the last insulin collection point of theprevious low glucose phase at 30 minutes (Immediate Stimulation), and(2) the highest insulin secretion in the high glucose phase to the lastinsulin collection point of the previous low glucose phase at 30 minutes(Maximum Stimulation). The SI indicates the magnitude of insulinreleased as stimulated by a higher concentration of glucose. Withcytokine exposure (+Ck), the SNM- and SμM-encapsulated islets underdiffusion (SNM,Diff & SμM, Diff) and the naked islet culture understatic conditions (Control) all showed a significant difference in themagnitude of insulin secreted upon high glucose challenge (ImmediateStimulation). The SNM-encapsulated islets under diffusion (SNM,Diff) andnaked islets cultured under static conditions (Control) showed anincrease in the magnitude of insulin secretion when the highest insulinsecretion in the high glucose phase was used (Maximum Stimulation).(Mean±SEM, n≥3, *p<0.05). FIG. 27C shows the shut-down index (SDI) wasthe ratio of (1) the first insulin collection point in the subsequentlow glucose phase at 70 minutes to the last insulin collection point inthe high glucose phase at 60 minutes (Immediate Shutdown), and (2) thelowest insulin secretion in the subsequent low glucose phase to the lastinsulin collection point in the high glucose phase at 60 minutes(Maximum Shutdown). The SDI reflects the magnitude of cessation ininsulin production once glucose concentration returns to normal. Withcytokine exposure (+Ck), the SμM-encapsulated islets under diffusion(SμM, Diff) exhibited the highest magnitude of insulin reductioncompared to the SNM-encapsulated islets under diffusion (SNM, Diff) andnaked islet culture (Control) as glucose dropped low (Immediate Shutdown& Maximum Shutdown). (Mean±SEM, n≥3, *p<0.05).

FIGS. 28A-B show in-vitro viability of mouse islets. FIG. 28A showsviability of mouse islets was measured following the 90-minutelow-high-low (1.6 mM, 16.6 mM, 1.6 mM) glucose stimulation in whichislets were subjected to the mock-loop circuit with (+Ck) or withoutcytokine exposure for SNM- and SμM-encapsulation under diffusion (SNM, D& SμM, D). The naked islet culture under static culture with cytokineexposure (Control, +Ck) showed significantly less viability compared toall other conditions. (Mean±SEM, n≥3, *p<0.05). FIG. 28B shows viable(green) and dead (red) cells were stained for control static culturewithout cytokines (A: Control), control static culture with cytokines(B: Control, +Ck), SNM-encapsulated mouse islets under diffusion withoutcytokines (C: SNM, D), SNM-encapsulated mouse islets under diffusionwith cytokines (D: SNM, D, +Ck), SμM-encapsulated mouse islets underdiffusion without cytokines (E: SμM, D), and SμM-encapsulated mouseislets under diffusion with cytokines (F: SμM, D, +Ck). Experiments withcytokine exposure (indicated by +Ck) consisted of media containingTNF-α, IFN-γ, and IL-1β. The control static culture with cytokines (B:Control, +Ck) showed significant level of islet damage compared to allother conditions.

FIG. 29 shows the rate of change in insulin secretion as depicted in thetable. The rate of change in insulin production was calculated based onthe slopes of curves that were fitted on glucose-insulin kinetics graphsto describe the quickness of insulin being secreted as glucoseconcentration changes. The SμM-encapsulated mouse islets under diffusionwithout cytokine exposure (SμM, Diff) showed similar rate of insulinsecretion in glucose-induced stimulation and a slightly faster insulincessation compared with the SμM-encapsulated mouse islets underdiffusion with cytokine exposure (SμM, Diff, +Ck).

FIGS. 30A-B shows SEM images of the pore-containing regions surroundedby solid silicon regions. FIG. 30A shows a top view SEM imageillustrating the rectangular pore-containing regions surrounded by solidsilicon regions, which provide mechanical support. FIG. 30B shows afurther magnified top view SEM image of the pore region showingindividual 10 nm-wide pores.

Example 3

Diffusion-based bioartificial pancreas (BAP) devices are limited by poorislet viability and functionality due to inadequate mass transferresulting in islet hypoxia and delayed glucose-insulin kinetics. Whileintravascular ultrafiltration-based BAP devices possess enhancedglucose-insulin kinetics, the polymer membranes used in these devicesprovide inadequate ultrafiltrate flow rates and result in excessivethrombosis. Here, the silicon nanopore membrane (SNM) exhibits a greaterhydraulic permeability and a superior pore size selectivity compared topolymer membranes for use in BAP applications. Specifically, theSNM-based intravascular BAP with ˜10 and ˜40 nm pore sized membranessupport high islet viability (>60%) and functionality (<15 minuteinsulin response to glucose stimulation) at clinically relevant isletdensities (5,700 and 11,400 IE/cm²) under convection in vitro. In vivostudies with ˜10 nm pore sized SNM in a porcine model showed high isletviability (>85%) at clinically relevant islet density (5,700 IE/cm²),c-peptide concentration of 144 pM in the outflow ultrafiltrate, andhemocompatibility under convection.

Materials and Methods

Silicon Nanopore Membranes (SNM) Architecture and Fabrication

Silicon nanopore membranes (SNM) have been prototyped from siliconsubstrates by MEMS technology as previously reported²⁶. Briefly, theprocess used the growth of a thin SiO₂ (oxide) layer on 400 μm-thickdouble side polished (DSP) silicon wafers followed by a low pressurechemical vapor deposition (LPCVD) of polysilicon (˜500 nm). The waferswere then specifically patterned, dry oxidized, wet etched, depositedwith a second polysilicon layer, and finally blanket-etched until 400 nmof polysilicon remained and the underlying vertical oxide layer wasexposed. The vertical sacrificial oxide layer defined the criticalnanoscale pore size of the membranes. The low temperature oxide (LTO)(˜1 μm) was deposited onto polysilicon of the wafers to serve as thehard mask for membrane protection. Deep reactive ion etching (DRIE)removed the backside of each window until membranes were disclosed.Eventually, the sacrificial oxide was etched away in 49% hydrofluoricacid (HF) during the final step of the fabrication process to leavebehind open nanoscale slit pores. The wafers were subsequently cut into1×1 cm chips with an effective area of 6×6 mm² containing 1500 windowseach, with a total of 10⁶ pores per membrane. Each rectangular pore was300 nm in depth and 2 μm in length. The SNM with an average pore sizewidth of ˜10 nm and ˜40 nm were used in this study. All membranes werecleaned using a conventional “piranha” clean procedure, which involved a20 min-immersion in 3:1 sulfuric acid (H₂SO₄)/hydrogen peroxide (H₂O₂)mixture, followed by thorough rinses in deionized (DI) water. Images ofSNM were obtained using scanning electron microscope (SEM) (Leo 1550)(FIGS. 1A, 30A, and 30B).

Surface Modification of SNM with poly(ethylene glycol) (PEG)

SNM were covalently modified with PEG using a previously reportedprotocol to prevent protein fouling on the membrane surface²⁶. Thetechnique used for PEG attachment involved a single reaction step whichcovalently couples silicon surface silanol group (Si—OH) to a chain ofPEG polymer through a trimethoxysilane group forming a Si—O—Si—PEGsequence. Briefly, SNM were immersed in a solution of 3 mM2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEG-silane) (Gelest:SIM6492.7) in toluene for 2 hr at 70° C. A series of extensive washingsteps involving toluene, ethanol, and DI water was used to removeunbounded PEG residue.

Hydraulic Permeability for SNM Pore Size Characterization

An automated mass and pressure measurement system was utilized forcharacterizing liquid flow through the SNM under a tangential-flowfiltration operation. The pore size of the SNM can be related tofiltration flow parameters using

$\begin{matrix}{{h = \sqrt[3]{\frac{12\mu\; l\; Q}{{{nw}\;\Delta\; P}\;}}},} & \left( {{Eq}.\mspace{14mu} 1} \right)\end{matrix}$where h is pore width, μ is the viscosity, l is the membrane thickness,Q is the volumetric flow rate, n is the number of pores per membrane, wis the pore length, and ΔP is the transmembrane pressure. To assemblethe overall system for SNM pore size characterization, air was appliedthrough a syringe pump (Sigma: Z675709) into a water reservoir. Waterwas circulated by a peristaltic pump (Masterflex: 07551-00) through adifferential pressure transducer (Omega: PX429 015GI), a flow cell withenclosed membrane, and returned to the original water reservoir. Theflow cell was assembled with the SNM submerged under water to remove airbubbles from all compartments. Specifically, a membrane was positionedwith the polysilicon interface facing down with a customized siliconegasket positioned on top of the membrane, followed by the finalplacement of a filtrate chamber on top of the gasket. All sections werefastened together and secured to the base with hand-tightened hex boltsuntil the gasket was visibly compressed. The ultrafiltrate permeatedthrough the membrane and was routed to a liquid collection containerthat rested on a precision mass balance (Mettler Toledo: XS205).Measurements from the differential pressure transducer and the massbalance were automatically collected with a data acquisition laptop. Atypical membrane hydraulic permeability test consisted of 5 ml/min flowrate and 4 pressure cycles (5, 1, 5, and 1 psi) for durations of 150 seach. Using the specifications for pore length, membrane thickness, andtotal number of pores provided based on individual wafer designs, theaverage pore size of SNM was calculated using Equation 1. All SNMmembranes in this study were surface-modified with PEG and exhibited anaverage pore size of ˜10 nm and ˜40 nm.Development of the Islet Chamber (IC)

In this study, IC possessed a thickness of 1000 μm and high isletdensities of 10% (5,700 IE/cm²) and 20% (11,400 IE/cm²). Islet densityby percentage was calculated as the ratio of total islet volumeexpressed in islet equivalents and the IC volume. Islet density bysurface area was calculated by dividing the total number of isletequivalents (IE) by the device membrane surface area. A biocompatibleacrylic sheet (McMaster: 8589K11) was first laser-cut to create ˜2.4mm×˜2.4 mm×˜1 mm thick void region which was inserted with eight 100 μmdiameter polytetrafluoroethlene (PTFE) coated wires (McMaster: 1749T11).A 2% agarose-islet mixture was then poured into this void region ofacrylic sheets. After the agarose-islet mixture was cured, all wireswere removed (FIG. 20D). Using this process, a hexagonal arrangement ofeight 800-μm cylindrical agarose-islet regions (dotted red circle) witha central 100 μm cylindrical channel (solid red circles) was obtainedfor the IC (FIG. 31). This configuration created a diffusion distance≤400 μm between the islets and ultrafiltrate. After IC construction, itwas assembled in the iBAP as described in FIGS. 21-22A with gasketsbetween the various iBAP components.

Assembly of an Intravascular Bioartificial Pancreas Device (iBAP) forIslet Encapsulation

The intravascular bioartificial pancreas device (iBAP) is shown in anexploded view in FIGS. 21-22A: the polycarbonate flow path componentcontaining the blood flow path, two SNM sandwiching the islet chamber(IC) containing the agarose (Sigma: A2576)-seeded mouse islets, thepolycarbonate backside (PC Backside), and the ultrafiltrate port(Ultrafiltrate Outlet). The parallel-plate blood flow path was modeledwith SolidWorks and computational fluid dynamics (CFD) to create idealflow characteristics to minimize thrombosis. The iBAP was symmetrical onboth sides and could be assembled with one IC on each side. The iBAP canpossess up to 0.72 cm² of SNM area. In operation, fluid flows throughthe Flow Path component at an elevated pressure creating a transmembranepressure (TMP) of ˜80 mmHg between the blood flow path and theUltrafiltrate Outlet resulting in ultrafiltrate flow through the SNM,IC, PC Backside, and Ultrafiltrate Outlet, which was collected in a tubein vitro or drained into interstitial tissue space in vivo. Under thediffusive condition, the PC Backside was capped-off resulting in noultrafiltrate flow through the system. All device components wereindividually sterilized either by autoclave or Nolvasan for both invitro and in vivo experiments.

Testing of the Intravascular Bioartificial Pancreas Device (iBAP) invitro

All procedures involving isolation of mouse islets were performed inaccordance with protocols approved by the Institutional Animal Care andUse Committee (IACUC) at the University of California, San Francisco(UCSF). Mouse islets were isolated from 8 to 10-week-old male B6 mice(Jackson Laboratories) based on previously described protocols.Harvested islets were maintained in suspension culture with RPMI 1640with L-glutamine and 11.1 mM glucose (Gibco: 11875-093), 10% fetalbovine serum (FBS) (Gibco: 16000), and 1% penicillin-streptomycin (P/S)(UCSF Cell Culture Facility: CCFGK003). A 2% agarose gel mixed withappropriate amount of mouse islets was dispensed into the previouslydescribed IC to create high islet densities of 10% or 20% by volume,respectively. The SNM with ˜10 nm and ˜40 nm pore sizes were chosen toencapsulate the IC with 10% and 20% islet density. For in vitroviability tests, a mock-loop circuit was set up with a peristaltic pumpflowing culture medium through the iBAP at TMP of ˜80 mmHg to generateultrafiltrate for convective condition (FIG. 22A), whereas noultrafiltrate was produced for the diffusive condition. The viabilityexperiments studied both ˜10 nm and ˜40 nm SNM encapsulating both 10%and 20% islet densities. After 3 days of culture, the devices weredisassembled and the islets were assessed for viability. Using this samemock-loop circuit, glucose-insulin kinetics was explored in iBAPscontaining either 10% or 20% islet density and either ˜10 nm or ˜40 nmpore sized SNM. SNM-encapsulated mouse islets in the iBAP were exposedto a low, high, and low glucose (Gibco: 11879) challenge on day 0.Ultrafiltrate directly produced from the IC under convection wascollected for insulin measurements. Insulin content was analyzed withmouse insulin enzyme-linked immunosorbent assay (ELISA) kits (Mercodia:10-1247-01) with accounted dilutions.

Implantation of the Intravascular Bioartificial Pancreas Device (iBAP)in Pigs

A preliminary proof-of-concept study was designed with a swine modelbecause of the comparably sized vasculature and hematologic similaritieswith humans. The study was approved by the IACUC review committee at PMIPreclinical CRO, San Carlos, Calif.

For pig #1, the device was assembled as previously described with eachchamber containing a 5% islet equivalents (IE) density by volume ofmouse islets suspended in agarose gel. Oral aspirin (81 mg) andclopidogrel (75 mg) were given to a 75 kg female Yorkshire pig for 3days preoperatively and then daily thereafter. After general anesthesiawas induced, a vertical incision was made to the left of midline toexpose the left external jugular vein. A 15Fr double-lumen tunneledcatheter (NextStep®, Teleflex, Morrisville, N.C.) was placed in the leftexternal jugular vein for blood sampling. The right carotid artery andright external jugular vein were then exposed via a similar verticalincision on the right side of the neck. Once the vessels were exposed asubcutaneous pocket was created caudally for eventual device placement.Heparin was given intraoperatively targeting an activated clotting time(ACT) of greater than 200 seconds. The 6 mm externally-supportedpolytetrafluoroethylene (PTFE) grafts were then anastomosed end-to-sideto the internal carotid artery for inflow and the external jugular veinfor outflow. The device was then placed in the subcutaneous pocket andanchored to surrounding soft tissue. The inflow and outflow grafts werethen connected to the device and clamps were removed to allow blood flowthrough the device, which was visually confirmed (FIGS. 34A-D). Theoverlying soft tissue and skin were then closed in layers and thenanimal was extubated and allowed to recover. Meloxicam and buprenorphinewere administered as needed for post-operative pain.

For pig #2, the device was assembled as previously described with 10%density by volume for each chamber. One chamber had a channel incommunication with the islets that was open to atmosphere, allowing forultrafiltrate flow through the chamber (FIG. 22A). The ultrafiltratethen passed through the channel and was freely deposited into thesurrounding tissue and subcutaneous pocket (FIG. 35A). The otherchamber's ultrafiltrate outlet was capped resulting in a diffusivechamber. The technical aspects of the implant procedure were identicalto Pig #1. Blood flow through the device and ultrafiltrate depositioninto the surrounding tissue was visually confirmed prior to closure ofthe incision.

Assessment of Islet Function in vivo

Islet function was assessed using standard intravenous glucose tolerancetests (IVGTT) with administration of glucose (0.5 g/kg in 40% solution)via the tunneled venous catheter. Blood was drawn to measure serumglucose using a standard glucometer (Accu-Chek Compact Plus: 1002-5021)at time 0, 5, 10, 15, 30, 60 and 90 minutes. The IVGTT was administeredon post-operative day (POD) 1 and 2 prior to the animals eating theirmorning meals. On POD 3 the test was performed intra-operatively priorto planned explant of the device and islet retrieval. For pig #1, bloodsampling for the intra-operative IVGTT was performed via directcannulation of the external jugular venous outflow tract immediatelydistal to the anastomosis. All samples from systematic circulation anddirectly collected from ultrafiltration port were stored on ice prior totesting for mouse insulin enzyme-linked immunosorbent assay (ELISA)(Mercodia: 10-1247-01) and c-peptide ELISA kits (EMD Millipore:EZRMCP2-21K).

Patency Assessment and Device Explant with Islet Retrieval

On POD 3 both animals were taken back to the operating room forassessment of patency and device retrieval. Once the animal wasintubated and sedated the incision was re-opened and the device wasdelivered into the superficial tissue for visual assessment andconfirmation of maintained patency. A final IVGTT was administered. Asmentioned, for pig #1, blood was sampled directly from the outflow veinvia direct cannulation of the external jugular vein distal to theanastomosis. For pig #2, blood was sampled from the tunneled catheter.Once the IVGTT was completed, the carotid was cannulated proximal to theanastomosis with a 5Fr catheter. Radiopaque contrast media was theninjected (Visipaque™, GE Healthcare, Little Chalfont, United Kingdom) tofluoroscopically confirm flow through the device (FIGS. 34A & 35A). Theinflow and outflow grafts were then clamped and the device was thenexplanted and subsequently flushed with culture media prior todisassembly and retrieval of the islets from the chamber.

Islet Viability

Islet viability was assessed by double staining with the Live/Dead CellImaging Kit (488/570) (Life Technology: R37601). Live cells aredistinguished by the presence of intensely fluorescent calcein (green)which is well-retained within live cells, whereas dead cells are stainedwith red. Briefly, agarose-encapsulated mouse islets were incubated inthe mixture of live (green) and dead (red) kit components for 15 min andextensively washed in phosphate buffered saline (PBS) to remove excessstaining. Images of mouse islets were obtained using laser scanningNikon Spectral Clsi confocal microscope (Nikon Instruments). Thepercentage of viability was calculated based on the ratio of non-dead orthe green area over the entire area of that islet.

Explanted Membrane Analysis

For observation, SNM were fixed in a solution containing 3%glutaraldehyde (Sigma: G7651), 1 M sodium cacodylate (Polysciences) and0.1 M sucrose (Sigma). After 2 days, the substrates were washed withdistilled water. Dehydration was achieved by placing these scaffolds inan increasing concentration of ethanol (50-100%). Dehydrated sampleswere then mounted on aluminum stubs, sputter-coated with gold-palladium,and examined with scanning electron microscopy (SEM) (Ultra 55, CarlZeiss).

Blood Platelet Adhesion and Activation

The SNM were fixed with 4% paraformaldehyde followed by PBS washes andincubated in blocking solution (PBS, 1% bovine serum albumin (BSA)) for30 min. Samples were then incubated with CD41 antibody (Biorbyt:orb181793) for platelet adhesion (green) and CD62p antibody (Bioss:bs-0561R-Cy3) for platelet activation (red) at a dilution of 1:300 for 4h and repeatedly washed with PBS to remove residues. Images wereobtained using 6D High Throughput Perfect Focus System (NikonInstruments).

Statistical Analysis

Sample pairs were analyzed using Student's t-test. Multiple samples wereevaluated with one-way or two-way analysis of variance (ANOVA) followedby Bonferroni and multiple comparison using Graphpad Prism software (SanDiego, Calif.). A p value of <0.05 was accepted as statisticallysignificant for all analyses.

Results

iBAP Testing in vitro

The iBAP comprising a ˜10 nm pore sized SNM with 10% (5,700 IE/cm²) or20% (11,400 IE/cm²) mouse islet densities was investigated forglucose-stimulated insulin response and viability of the encapsulatedislets after three days. Under convection, the iBAP with 10% mouse isletdensity and ˜10 nm pore sized SNM showed an increase in insulinsecretion within 10 minutes of high glucose exposure (FIG. 32A (i)),which was consistent with normal islet function of biphasic insulinrelease (i.e. the first insulin phase appeared within 5-10 minutesfollowed by a second sustained phase that is slower and delayed as timesgoes longer). Furthermore, during the period of 63 to 78 minutes inwhich the glucose concentration decreased, the correspondingstimulated-insulin secretion also dropped. However, when the celldensity increased from 10% to 20% in the iBAP with ˜10 nm pore sized SNMunder convection, no significant change in glucose-stimulated insulinlevel was observed within the first few minutes of high glucose exposure(FIG. 32A (ii)). The stimulation index (SI), the ratio of stimulated tobasal insulin secretion normalized by the insulin content, wascalculated as 4.4±0.6 and 1.1±0.1 for the iBAP of ˜10 nm pore sized SNMwith 10% and 20% mouse islet densities, respectively. It iswell-recognized that delay of insulin secretion in response to glucose(>15 min) has been a common problem encountered in the earlyextravascular hollow-fiber systems. Our iBAP with ˜10 nm pore size underconvection supported the normal insulin function at 10% islet densitywith no significant delay in glucose-stimulated insulin response.However, the glucose stimulated-insulin response at 20% islet densityunder convection with ˜10 nm pore sized SNM showed abnormalinsulin-functioning behavior, indicating that encased cells were likelynot in optimal health in that environment.

The viability study of ˜10 nm pore sized SNM in the iBAP demonstratedthat 10% mouse islet density under convection (40±11%) showed a higherviability compared to that under diffusion (4.0±1.3%) (FIGS. 32B-C).Furthermore, as the islet density increased to 20% within the isletchamber, the viability significantly decreased under diffusion (11±5.8%)and convection (17±11%) (FIGS. 32B-C). In summary, the ˜10 nm pore sizedSNM under convection is sufficient to support the viability andglucose-insulin response of 10% (but not 20%) mouse islet density.

To verify whether the pore size was the limiting factor in causing celldeath at the higher density, the ˜40 nm pore sized SNM with 10% or 20%mouse islet densities was studied in the iBAP under diffusion andconvection. The glucose-stimulated insulin study showed that both 10%(FIG. 33A (i)) and 20% (FIG. 33A (ii)) demonstrated the characteristicinsulin biphasic release curves. The iBAP at 10% and 20% mouse isletdensities with ˜40 nm pore sized SNM indicated that the first spike inglucose-stimulated insulin production occurred within 10 minutes of highglucose exposure. Both conditions showed insulin shut down as glucoseconcentration decreased. The SI at 10% and 20% mouse islet densitieswith ˜40 nm pore sized SNM were 3.2±1.3 and 9.1±1.2, respectively.Although the absolute amount of insulin secreted did not double whencell density increased from 10% to 20%, the latter showed a 1.9-foldincrease in SI factor, indicating the magnitude of insulin stimulatedfrom basal to high glucose level almost doubled. The viability studydemonstrated that 10% mouse islet density under convection (66±4.8%)showed a higher viability compared to that under diffusion (24±6.8%)(FIGS. 33B-C). Furthermore, as the islet density increased to 20% withinthe islet chamber, the viability of islets under convection (61±3.0%)exhibited a significant increase in viability compared with that underdiffusion (5.2±1.3%) (FIGS. 33B-C). Overall, the iBAP using ˜40 nm poresized SNM under convection supported the viability and glucose-insulinresponse at both 10% and 20% mouse islet densities.

Compared to the previous in vitro experiments using ˜10 nm pore sizedSNM, the ˜40 nm pore sized SNM enhanced the viability at 10% and 20%mouse islet densities to 66±4.8% and 61±3.0% as compared to 40±11% and17±11% for the ˜10 nm pore sized SNM under convection, respectively(FIGS. 32D and 33B). Islet viability correlates positively with anincrease in pore size dimension under convection. The greater amount ofultrafiltrate produced by ˜40 nm pore size under convection enhanced theviability and functionality of encapsulated islets. In contrast,diffusion provides inadequate mass transfer to support a greater isletdensity. Although an increase in pore size improved islet viability at alower cell density (10%) under diffusion from ˜10 nm (4.0±1.3%) to ˜40nm pore size (24±6.8%), the islet viability at a higher cell density(20%) showed no significant difference under diffusion ((11±5.8%) vs.(5.2±1.3%)). These data show that nutrients and oxygen remained severelydepleted under diffusion even when the pore size was increased to ˜40nm. Diffusive mass transport has been widely reported for porousmaterials with nanometer-sized pores, as one study showed that thediffusion of 45 nm nanoparticles was slowed down by a factor of 2 in 300nm cylindrical pores due to hydrodynamic friction. Therefore, given thegreater cell density, the large diffusion distance, and the restrictionof nanoscaled pores under diffusion, insufficient transfer of nutrientsand oxygen would likely result in cell necrosis and hypoxia. In summary,our in vitro testing of the iBAP demonstrated that convection is the keyto supporting 10% or 20% mouse islet density with either ˜10 nm or ˜40nm pore sized SNM with higher islet viability and providing appropriateglucose-stimulated insulin response.

iBAP Implantation in Pigs

As a first step to study the device and membrane patency, thediffusion-based iBAP with ˜10 nm pore sized SNM and a 5% mouse isletdensity (2,850 IE/cm²) was intravascularly grafted in the porcine modelfor three days. The angiogram showed no thrombosis formation andobstruction in the blood flow path of the device during explant (FIG.34A (i)). This data matched with previous studies in which the iBAPdevice was intravascularly implanted into Class A dogs where the devicewas patent throughout the experiment, possessed no thrombus formation,and generated 27.5 ml of ultrafiltrate based on a SNM pore size of 5.6nm after explanted at 8 days. A cytokine panel indicated an expectedincrease in the pro-inflammatory response from pig immediately after thesurgery (FIG. 37). SEM images of blood-contacting SNM displayed somenon-catastrophic attachment and aggregations of cells and adhesiveproteins (FIG. 34A (ii)). In particular, red blood cells, white bloodcells, and platelets were deposited on the membrane surface. Subsequentimmunohistochemical analysis showed that while platelet adhesion mostlyin the porous regions of SNM (which contain the nanopores) as indicatedby the green CD41 marker, there was minimal platelet activation observedas stained by the red CD62p marker (FIG. 34B). The viability studydemonstrated that the diffusion-based iBAP in the pig supported theviability at 5% mouse islet density with ˜10 nm pore size (88±4.9%)which was comparable with the in vitro conditions (89±2.1%) (FIG. 34C).To avoid hypoxia and necrosis of cells located at the center ofdiffusion-based devices, islet density of the macrocapsules has beensuggested to be 5-10% of the volume fraction in order to ensure theproper exchange of nutrients and waste of islets. Our iBAP with ˜10 nmpore sized SNM demonstrated sufficient mass transfer to support theviability of 5% islet density under diffusion (FIG. 34D). Unfortunately,the concentration of mouse insulin and c-peptide in porcine systemiccirculation was below the detection limit.

Next, the performance of the iBAP with SNM-encapsulated mouse isletsunder diffusion and convection at a higher islet density was evaluatedto demonstrate the effectiveness of convective mass transfer forsupporting islet viability and functionality. Specifically, the iBAPwith ˜10 nm pore size SNM and a 10% mouse islet density (5,700 IE/cm²)with convective and diffusive mechanism was grafted to the carotidartery and vein of a pig for three days. A pro-inflammatory response wasalso observed for this pig immediately after the surgery (FIG. 38). Noultrafiltration was generated for the diffusive side, whereasultrafiltrate production was observed on the convective side. Theultrafiltrate was directly drained into the interstitial space of theanimal. After three days, no significant change in device blood flowrate and the ultrafiltrate appeared to be clear, indicating themembranes were intact during the in vivo experiment (FIG. 35A (i)). Theangiogram also showed no thrombus formation and obstruction in the bloodflow path of the device during explant. Gross inspection of theblood-contacting membrane surfaces showed minimal cellular adhesion forboth diffusive and convective conditions; however, the back side of theSNM under convection exhibited a white layer of proteinaceous materials(FIG. 35B (iii)). Our previous study showed that a pore size of ˜7 nmSNM can prevent the passage of large molecules such as bovine serumalbumin (66.5 kDa). SEM images of blood-contacting SNM displayed minimalcellular attachment for the diffusive case, whereas there appeared to bemore cellular deposition were present for the convective condition (FIG.35A (ii)). Subsequent immunohistochemical analysis showed that theconvection resulted in more platelet adhesion and activation on theblood-exposed SNM surface compared with diffusion (FIG. 35B). Moreimportantly, the viability of 10% mouse islet density with ˜10 nm poresize was higher in the convective condition (85±4.4%) compared to thediffusive scenario (73±4.1%). Interestingly, the in vivo viability at10% mouse islet density with ˜10 nm pore size under diffusion (73±4.1%)was greater than the in vitro viability of those under diffusion(2.0±1.3%) and convection (40±11%). The ultrafiltrate generated directlyfrom the islet chamber on the convective side indicated a mousec-peptide concentration of 144 pM (or 12 pg/min/IE (islet equivalent)insulin production rate), exhibiting the functionality of theencapsulated islets. These data demonstrate that SNM encapsulation underconvection preserved islet viability and functionality of theencapsulated cells at a cell density for macroencapsulation. Tosummarize, the in vitro testing of the iBAP demonstrated that SNM with˜10 nm pore size showed an improved viability at 10% mouse islet density(5,700 IE/cm²) under convection, and SNM with ˜40 nm pore sizedemonstrated an increase in viability at 10% (5,700 IE/cm²) and 20%(11,400 IE/cm²) mouse islet densities compared to those tested underdiffusion. Furthermore, the glucose-insulin kinetics experiments showedphysiological glucose-insulin response and a clinically relevantabsolute insulin production rate. Furthermore, porcine studiesdemonstrated both device and membrane patency under convection anddiffusion, a higher islet viability at 10% mouse islet density withconvection, and a clinically relevant mouse insulin production rate on aper IE basis. Overall, these studies show the feasibility of designing afull-scale SNM-based iBAP to achieve long-term blood flow patency,improved islet viability with convection in comparison to diffusion atclinically relevant densities, and sustained clinically relevant insulinsecretion on a per IE basis.

Example 4

The silicon nanopore membrane possesses ultra-high-hydraulicpermeability. A range of different pore-size SNM (5-500 nm) has beentested to generate the appropriate ultrafiltrate rates to deliver thenecessary convective mass transfer of nutrients and insulin, while stillmaintaining immunoisolation. FIG. 39 presents hydraulic permeabilitydata for various pore-size SNM and FIGS. 30A-B present scanning electronmicroscopy (SEM) images of 10 nm-wide SNM.

The silicon nanopore membrane possesses ultra-selective preciseslit-shaped nanopores to achieve immunoisolation.

SNM with highly precise pore were fabricated using an innovative processand demonstrated excellent hydraulic permeability.Microelectromechanical systems (MEMS) fabrication technology producedthe SNM. A thin sacrificial SiO₂ layer is grown defining the submicronpore size of the membrane. Thermal oxidation of silicon substratesprovides oxides down to 3 nm in thickness with <1% variation. The oxideis etched leaving behind open parallel-plate nanochannel pores.0.25-1.00 μm-thick polycrystalline silicon membranes with pore sizesbetween 5-500 nm supported by a 400 μm-thick support structure werefabricated. The SNM hydraulic permeability was tested in a custom flowcell with cross-flow and a transmembrane (TMP) pressure. Hydraulicpermeability experiments demonstrated greater hydraulic permeability ofSNM than conventional polymer membranes.

Polymer coated SNM reduced protein adsorption and provided ahemocompatible surface. Polymer coatings were evaluated on siliconsubstrates to retard protein fouling. Polyethylene glycol (PEG),polysulfobetaine methyacrylate (pSBMA), and poly(N-vinyldextranaldonamide-co-N-vinylhexanamide) (PVAm-Dex/Hex) were chosen to reducenonspecific protein adsorption. A Rapid In Vivo IntraVascular Evaluation(RIVIVE) protocol inserted PEG-coated and uncoated (bare) silicon dartsin femoral veins of Wistar rats. After 30 days of implantation, thedarts and vessels were explanted and evaluated by gross examination,histology, and scanning electron microscopy (SEM) (FIG. 40). Alluncoated silicon darts had adherent platelet-fibrin clots, while thePEG-coated darts showed no adherent clot.

SNM demonstrated successful immunoisolation. Islet immunoisolation wasalso studied in vitro using cell culture medium with or withoutcytokines (IL-1β, TNF-α, and IFN-γ). Islets were encapsulated in betweentwo 7 nm pore SNM. After six hours, the islets were removed and stainedfor viability. Control static culture experiments were performed todetermine the effect of SNM. FIG. 41 indicates the ability of the 7 nmpore SNM to achieve islet immunoisolation. The large size of complementproteins (such as C4, 210 kDa, and C1q, 410 kDa) suggests limitedtransport through SNM. Complement permeation through SNM was evaluatedby a functional hemolytic assay measuring total complement activity asthe capacity of serum to lyse sheep red blood cells coated withanti-sheep erythrocyte antibodies. 100% of the total complement activitywas detected in the receiving chamber when commercially availablemembranes (100-400 nm pores) separated the two chambers. In contrast,less than 1% of total complement activity was detected with SNM andtrack-etch membranes. These results demonstrate SNM block largecomplement molecules.

A small-scale cell scaffold has been developed and enables testing ofenriched insulin producing cells under convection. In order to testultrafiltrate formation under convective mass transport, a small-scaleCell Scaffold has been developed and tested with islets. The CellScaffold consists of a hexagonal arrangement of eight 100 μm diametercylindrical ultrafiltrate channels (solid circles in FIG. 31) moldedinto a 2% agarose gel by eight 100 μm diameter PTFE coated wires tominimize the diffusion distance between the cells and ultrafiltrate.Biocompatible acrylic sheets were laser cut to create ˜2.4 mm×˜2.4 mm×˜1mm thick void region holding the cells, agarose, and ultrafiltratechannels in between SNM. FIGS. 20A-D illustrate the processes andfixtures for creating the Cell Scaffold, and FIG. 31 demonstrates theassembled Cell Scaffold: Cell Scaffold's acrylic sheet containing islets(white spheres), agarose, and cylindrical ultrafiltrate channels (solidred circles). The hexagonal arrangement of cylindrical channels createseight 800-μm cylindrical cell agarose tissue regions (dotted red circle)with a central 100 μm cylindrical channel. This configuration creates adiffusion distance ≤400 μm between the cells and ultrafiltrate.

A small-scale iBAP prototype has been developed for vitro and in vivoCell Scaffold testing and in vivo hemocompatibility testing. FIGS. 21and 22A describe the small-scale iBAP, which possesses up to 2 cm² ofSNM area. FIG. 21 is an exploded view of the iBAP components: thepolycarbonate Flow Path component containing the blood flow path, twoSNM, the Cell Scaffold containing the agarose seeded cells, thePolycarbonate Backside (PC Backside), and the Ultrafiltrate Outlet, Cellculture medium or blood flows through the Flow Path component at ˜80mmHg generating a TMP pressure between blood or cell culture medium andthe Ultrafiltrate Outlet resulting in ultrafiltrate flow through theSNM, Cell Scaffold, PC Backside, and Ultrafiltrate Outlet, which iscollected in a vein in the clinical setting or a collection tube invitro. FIG. 22A is a picture of the assembled small-scale iBAP used forin vitro and in vivo testing.

In vitro small-scale Cell Scaffold testing demonstrated increased isletviability with convection versus diffusion and promising glucose-insulinkinetics. Freshly isolated mouse islets at either 10% or 20% isletdensity by volume (or 5,700 IEQ/cm² or 11,400 IEQ/cm² respectively)within the small-scale Cell Scaffold were loaded into an iBAP containing40 nm SNM. The iBAP devices were connected to a mock circuit loop (FIG.42) in an incubator. For each islet density, three Cell Scaffolds weretested in diffusion or convection. After 3 days, the islets were stainedby FDA+PI to determine cell viability, FIG. 33C (iv and v) is arepresentative image from the 20% islet density experiments. For bothislet densities, Cell Scaffolds exposed to convection possessed greaterislet viability than Cell Scaffolds exposed to diffusion (FIG. 48). The40 nm SNM supported islet viability at clinically relevant isletdensities.

Human islet function was assessed in a 90-minute glucose-insulinkinetics study. Freshly isolated human islets at 10% islet density inthe small-scale Cell Scaffold were loaded into an iBAP with a 40 nm SNMand then stabilized in low glucose cell culture medium for 1 hour in themock circuit loop. At time zero, the glucose concentration was increasedfor 70 minutes and ultrafiltrate samples were collected from theUltrafiltrate Outlet. FIG. 43 demonstrates the first insulin peak at ˜8minutes followed by sustained and heightened insulin secretion until theglucose concentration was reduced at 70 minutes, where a decrease ininsulin production was then observed. A clinically functioning iBAP mustpossess an insulin response in <15 minutes to achieve effective glycemiccontrol.

A prototype full-scale iBAP was designed, CFD modeled, and demonstratedblood flow path patency in an in vivo 7-day hemocompatibility study.Full-scale iBAP blood flow path designs were generated in SolidWorks andanalyzed in ANSYS Fluent to determine the feasibility of extending theblood flow path from the small-scale iBAP prototype. This firstgeneration full-scale iBAP was manufactured from medical gradepolycarbonate and loaded with SNM. The full-scale iBAP wasintravascularly implanted into a healthy pig (FIG. 44) and heparin wasadministered at 100 units/kg perioperatively and then a twice-dailyregime of 1.5 mg/kg of acetylsalicylic acid (aspirin). The device wasexplanted at 7 days and was patent throughout the experiment.

Example 5

The iBAP will be connected to arterio-venous grafts and a pressure dropbetween the artery and vein will produce ultrafiltrate flow through theSNM encapsulated Cell Scaffold seeded with insulin producing cells,carrying nutrients to the cells and insulin to the ultrafiltrate vein(FIG. 36A-B). The SNM is a biocompatible and high hydraulic permeabilitymembrane that produces high levels of ultrafiltrate enabling physiologicnutrient delivery to, and insulin secretion from, the Cell Scaffold,while the cells are human embryonic stem cell (hESC) derived mature betacells arranged in ˜100 μm diameter spheres possessing glucose stimulatedinsulin secretion both in vitro and in vivo.

FIG. 31 shows a gross image of islets and agarose mixture inside the ICin which the maximum diameter surrounding each ultrafiltrate channel is800 μm. The figure shows a microscopic image of the Cell Scaffoldcontaining agarose gel, islets, and cylindrical ultrafiltrate channels.

FIGS. 32A-C shows in vitro testing of the intravascular bioartificialpancreas device (iBAP) with 10% or 20% islet density encapsulated with10 nm-pore size SNM. FIG. 32A shows glucose-insulin kinetics of theSNM-encapsulated iBAP with 10% (i) or 20% (ii) islet densities underconvection was measured from exposing them to a series of low, high, andlow glucose conditions. FIG. 32B shows the SNM-encapsulated iBAP with10% islet density under convection (10% convection) showed significantlyhigher viability compared to that of 10% islet density under diffusion(10% diffusion), and 20% islet density under both diffusion (20%diffusion) and convection (20% convection) after 3 days. (n>3, *p<0.05).Viabilities of islets that were immediately encapsulated in agarose anddispensed into the islet chamber (IC) without further testing wereevaluated as the in vitro positive control. FIG. 32C shows viable(green) and dead (red) cells were stained for in vitro positive control(i), 10% islet density under diffusion (ii), 10% islet density underconvection (iii), 20% islet density under diffusion (iv), and 20% isletdensity under convection (v) (scale bar=50 μm). The SNM-encapsulatediBAP with 10% islet density under convection (iii) showed higherviability than that of 10% islet density under diffusion (ii), and 20%islet density under both diffusion (iv) and convection (v). The 10%islet density under diffusion (ii), and 20% islet density under bothdiffusion (iv) and convection (v) showed similar viability withsignificant amount of cell death.

FIGS. 33A-C shows in vitro testing of the intravascular bioartificialpancreas device (iBAP) with 10% or 20% islet density encapsulated with40 nm-pore size SNM. FIG. 33A shows glucose-insulin kinetics of theSNM-encapsulated iBAP with 10% (i) or 20% (ii) islet densities underconvection was measured from exposing them to a series of low, high, andlow glucose conditions. FIG. 33B shows the SNM-encapsulated iBAP with10% and 20% islet density under convection (10% & 20% convection) showedsignificantly higher viability compared to that of 10% and 20% isletdensity under diffusion (10% & 20% diffusion) after 3 days (n>3,*p<0.05). Viabilities of islets that were immediately encapsulated inagarose and dispensed into the islet chamber (IC) without furthertesting were evaluated as the in vitro positive control. FIG. 33C showsviable (green) and dead (red) cells were stained for in vitro positivecontrol (i), 10% islet density under diffusion (ii), 10% islet densityunder convection (iii), 20% islet density under diffusion (iv), and 20%islet density under convection (v) (scale bar=50 μm). TheSNM-encapsulated iBAP with 10% and 20% islet density under convection(iii & v) showed higher viability than those under diffusion (ii & iv).In particular, the 20% islet density under diffusion (iv) showedsignificant amount of cell death.

FIGS. 34A-D shows in vivo testing of the intravascular bioartificialpancreas device (iBAP) with 5% islet density encapsulated with 10nm-pore size SNM for 3 days. FIG. 34A shows an image of the explanteddiffusion-based iBAP (i). An SEM image of the implanted membrane showingattachment of red blood cells and platelets (ii) (scale bar=10 μm). FIG.34B shows immunofluorescence staining of platelet adhesion CD41 marker(green) and platelet activation CD62p marker (red). The rectangularpore-containing regions surrounded by solid silicon regions were shownin the bright field image (i). The platelet adhesion (green) mostlyoccurred in the window regions where pores reside, whereas minimalplatelet activation (red) was detected (ii) (scale bar=20 μm). FIG. 34Cshows the SNM-encapsulated iBAP with 5% islet density under diffusionboth in vitro (in vitro 5% diffusion) and in vivo (in vivo 5% diffusion)showed significantly higher viability compared to the in vitro negativecontrol (n>3, *p<0.05). The in vitro negative control was those isletsthat were assembled in the iBAP with no medium circulation for 3 days.Viabilities of islets that were immediately encapsulated in agarose anddispensed into the islet chamber (IC) without further testing wereevaluated as the in vitro positive control. FIG. 34D shows viable(green) and dead (red) cells were stained for in vitro positive control(i), in vitro negative control (ii), in vitro 5% islet density underdiffusion (iii), in vivo 5% islet density under diffusion (iv) (scalebar=50 μm). The SNM-encapsulated iBAP with 5% islet density underconvection (iii & v) showed similar viability to the in vitro positivecontrol.

FIGS. 35A-D shows in vivo testing of the intravascular bioartificialpancreas device (iBAP) with 10% islet density encapsulated with 10nm-pore size SNM under either diffusion or convection for 3 days. FIG.35A shows an image of the explanted iBAP with diffusion (back) andconvection (front) of the device (i). An SEM image of the diffusion-sideimplanted membrane showed a patent surface (ii, top) (scale bar=100 μm)and an SEM image of the convection-side membrane presented coverage ofproteins and cells on the surface (ii) (scale bar=10 μm). FIG. 35B showsimmunofluorescence staining of platelet adhesion CD41 marker (green) andplatelet activation CD62p marker (red). The rectangular pore-containingregions surrounded by solid silicon regions were shown in the brightfield image for diffusion-side membrane (i) and convection-side membrane(iii). The platelet adhesion (green) was minimal on the diffusion-sidemembrane (ii), whereas more platelet adhesion (green) and activation(red) was detected on the convection-side membrane (iv) (scale bar=20μm). FIG. 35C shows the SNM-encapsulated iBAP with 10% islet densityunder convection both in vitro (in vitro 10% convection) and in vivo (invivo 10% convection) showed higher cell viability compared to that underdiffusion in vitro (in vitro 10% diffusion) and in vivo (in vivo 10%diffusion). (n>3, *p<0.05). The in vitro negative control was thoseislets that were assembled in the iBAP with no medium circulation for 3days. Viabilities of islets that were immediately encapsulated inagarose and dispensed into the islet chamber (IC) without furthertesting were evaluated as the in vitro positive control. FIG. 35D showsviable (green) and dead (red) cells were stained for in vitro positivecontrol (i), in vitro negative control (ii), in vitro 10% islet densityunder diffusion (iii), in vitro 10% islet density under convection (iv),in vivo 10% islet density under diffusion (v), in vivo 10% islet densityunder convection (vi) (scale bar=50 μm). The SNM-encapsulated iBAP with10% islet density under convection in vivo (vi) showed similar viabilityto the in vitro positive control.

FIGS. 36A-B shows blood flow in the iBAP. FIG. 36A shows an illustrationof the full-scale iBAP connected to arterial-venous grafts and anUltrafiltrate Outlet catheter delivering insulin rich ultrafiltrate tothe ultrafiltrate vein. Blood flows into the iBAP and a looped bloodchannel transports blood to a vein. The SNM encapsulated IC is placeddirectly above and below the blood channel. FIG. 36B shows across-sectional view perpendicular to blood flow illustrating the bloodchannel surrounded by the SNM (green) encapsulated IC (blue).Ultrafiltrate (black arrows) crosses the SNM encapsulated IC intoultrafiltrate channels (side) and exits the Ultrafiltrate Outletcatheter into the ultrafiltrate vein.

FIG. 37 shows daily measurement of the systematic cytokine concentrationin the pig. The intravascular bioartificial pancreas (iBAP) with 5%islet density encapsulated with 10 nm-pore size SNM. Cytokines namelygranulocyte-macrophage colony-stimulating factor (GM-CSF), tumornecrosis factor-alpha (TNF-α), interleukin 1-alpha (IL-1α), interleukin1-beta (IL-1β), interleukin 8 (IL-8), interleukin 12 (IL-12),interleukin 18 (IL-18), interleukin-1 receptor antagonist (IL-1Ra),interleukin 4 (IL-4), and interleukin 10 (IL-10) were analyzed.Interferon gamma (IFN-γ) was not detected. About 35.89 pg/ml ofinterleukin 2 (IL-2) was detected post-implantation on Day 0 only.

FIG. 38 shows daily measurement of the systematic cytokine concentrationin the pig. The intravascular bioartificial pancreas (iBAP) with 10%islet density encapsulated with 10 nm-pore size SNM. Cytokines namelyInterferon gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α),interleukin 1-alpha (IL-1α), interleukin 1-beta (IL-1β), interleukin 2(IL-2), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 18(IL-18), interleukin-1 receptor antagonist (IL-1Ra), interleukin 4(IL-4), and interleukin 10 (IL-10) were analyzed. Granulocyte-macrophagecolony-stimulating factor (GM-CSF) was not detected. About 25.44 pg/mlof interleukin 8 (IL-8) was detected on Day 2 only.

FIG. 39 shows silicon nanopore membrane (SNM) hydraulic permeability asa function of pore size.

FIG. 40 shows SEM images of uncoated (left) and PEG-coated (right)silicon surfaces at low (top) and high (bottom) magnification after 30days of blood exposure in vivo in femoral vessels of anticoagulant freerodents. The uncoated samples displayed adherent platelet-fibrin clots,while the coated surfaces were generally free of thrombus.

The invention claimed is:
 1. A bioartificial ultrafiltration devicecomprising: a planar scaffold comprising a matrix comprising: apopulation of cells and a plurality of channels adjacent to thepopulation of cells, wherein the channels extend from a first surface toa second surface of the planar scaffold and are substantiallyperpendicular to the first and second surface of the planar scaffold; afirst semipermeable ultrafiltration membrane disposed on the firstsurface of the planar scaffold; a first compartment adjacent to thefirst surface of the planar scaffold and in fluidic communication withthe planar scaffold via the first semipermeable ultrafiltration membraneand comprising an inlet and an outlet; a second semipermeableultrafiltration membrane disposed on the second surface of the planarscaffold; a second compartment adjacent to the second surface of theplanar scaffold and in fluidic communication with the planar scaffoldvia the second semipermeable ultrafiltration membrane and comprising anoutlet, wherein the first and the second semipermeable ultrafiltrationmembranes comprise a plurality of pores, wherein the plurality of poresof the first semipermeable ultrafiltration membrane are rectangularpores having a depth of 100-1000 nm, a width of 3 nm-50 nm and a lengthof 1 micron-5 micron, wherein the first semipermeable ultrafiltrationmembrane allows transport of ultrafiltrate from the first compartment tothe plurality of channels and wherein the ultrafiltrate traverses fromthe plurality of channels, across the second semipermeableultrafiltration membrane, into the second compartment.
 2. The device ofclaim 1, wherein the second semipermeable ultrafiltration membranecomprises a plurality of pores having a width larger than the width ofthe plurality of pores in the first semipermeable ultrafiltrationmembrane.
 3. The device of claim 1, wherein the inlet of the firstcompartment is attachable to a tubing for connection to a blood vesselof a subject.
 4. The device of claim 3, wherein the blood vessel is anartery of the subject.
 5. The device of claim 1, wherein the outlet ofthe first compartment is attachable to a tubing for connection to ablood vessel of a subject.
 6. The device of claim 5, wherein the outletof the first compartment is attachable to a tubing for connection to avein of the subject.
 7. The device of claim 5, wherein the outlet of thefirst compartment is attachable to a tubing for connection to an arteryof the subject.
 8. The device of claim 7, the artery connected to theoutlet is the same artery as connected to the inlet.
 9. The device ofclaim 1, wherein the first compartment comprises a plurality of outletsthat are each attachable to tubings for connection to (i) a plurality ofdifferent blood vessels of a subject or (ii) a plurality of connectionsites on a single blood vessel.
 10. The device of claim 1, wherein theoutlet of the second compartment is attachable to a tubing forconnection to a blood vessel of a subject.
 11. The device of claim 10,wherein the outlet of the second compartment provides the ultrafiltrateto one or more blood vessels of the subject.
 12. The device of claim 11,wherein the outlet of the second compartment is attachable to a tubingfor connection to one or more veins of the subject.
 13. The device ofclaim 11, wherein the outlet of the second compartment is attachable toa tubing for connection to one or more arteries of the subject.
 14. Thedevice of claim 11, wherein the outlet of the second compartment isattachable to an analyte analysis device.
 15. The device of claim 1,wherein the second compartment comprises a plurality of outlets forproviding the ultrafiltrate to at least one blood vessel of the subject.16. The device of claim 1, wherein the second compartment comprises aplurality of outlets for providing the ultrafiltrate to an analyteanalysis device.
 17. The device of claim 1, wherein the cells areinsulin producing cells.
 18. The device of claim 1, wherein the pores ofthe second semipermeable ultrafiltration membrane are rectangular poreshaving a depth of 100-1000 nm, a width of 3 nm-50 nm and a length of 1micron-5 micron.